U.S. patent number 7,635,722 [Application Number 09/744,622] was granted by the patent office on 2009-12-22 for chemical induced intracellular hyperthermia.
This patent grant is currently assigned to Saint Jude Pharmaceuticals, Inc.. Invention is credited to Nicholas Bachynsky, Woodie Roy.
United States Patent |
7,635,722 |
Bachynsky , et al. |
December 22, 2009 |
Chemical induced intracellular hyperthermia
Abstract
An invention relating to therapeutic pharmacological agents and
methods to chemically induce intracellular hyperthermia and/or free
radicals for the diagnosis and treatment of infections, malignancy
and other medical conditions. The invention relates to a process
and composition for the diagnosis or killing of cancer cells and
inactivation of susceptible bacterial, parasitic, fungal, and viral
pathogens by chemically generating heat, and/or free radicals
and/or hyperthermia-inducible immunogenic determinants by using
mitochondrial uncoupling agents, especially 2,4 dinitrophenol and,
their conjugates, either alone or in combination with other drugs,
hormones, cytokines and radiation.
Inventors: |
Bachynsky; Nicholas (Parkland,
FL), Roy; Woodie (Parkland, FL) |
Assignee: |
Saint Jude Pharmaceuticals,
Inc. (Texarkana, TX)
|
Family
ID: |
22244263 |
Appl.
No.: |
09/744,622 |
Filed: |
July 27, 1999 |
PCT
Filed: |
July 27, 1999 |
PCT No.: |
PCT/US99/16940 |
371(c)(1),(2),(4) Date: |
May 07, 2002 |
PCT
Pub. No.: |
WO00/06143 |
PCT
Pub. Date: |
February 10, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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60094286 |
Jul 27, 1998 |
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Current U.S.
Class: |
514/728;
424/176.1 |
Current CPC
Class: |
A61K
38/26 (20130101); A61K 41/0052 (20130101); A61K
31/277 (20130101); A61K 31/06 (20130101); A61K
45/06 (20130101); A61P 31/00 (20180101); A61P
33/00 (20180101); A61K 31/00 (20130101); A61K
31/06 (20130101); A61K 31/00 (20130101); A61K
31/06 (20130101); A61K 2300/00 (20130101); Y02A
50/30 (20180101); Y02A 50/409 (20180101); Y02A
50/401 (20180101); Y02A 50/411 (20180101) |
Current International
Class: |
A01N
33/18 (20060101); A01N 33/24 (20060101); A61K
31/045 (20060101); A61K 39/00 (20060101) |
Field of
Search: |
;514/728,12
;424/176.1 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO 97/05870 |
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Feb 1997 |
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WO |
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WO-97/35540 |
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Oct 1997 |
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WO |
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Primary Examiner: Marschel; Ardin
Assistant Examiner: Royds; Leslie A
Attorney, Agent or Firm: Fulbright & Jaworski LLP
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is the National Stage application of International
Application No. PCT/US99/16940 filed Jul. 27, 1999 which claims
benefit of priority to U.S. provisional patent application Ser. No.
60/094,286, filed Jul. 27, 1998, which is hereby incorporated by
reference in its entirety.
Claims
What is claimed is:
1. A method for inducing intracellular hyperthermia in a subject
comprising the step of administering to a subject having an
infection of Borrelia burgdorferi, Mycobacterium leprae, Treponema
pallidum, HIV, hepatitis C, or herpes virus, an amount of
2,4-dinitrophenol sufficient to induce whole body intracellular
hyperthermia in the subject, wherein the whole body intracellular
hyperthermia is sufficient to treat the Borrelia burgdorferi,
Mycobacterium leprae, Treponema pallidum, HIV, hepatitis C, or
herpes virus infection in the subject.
2. The method of claim 1, wherein a second medication is
administered, wherein the second medication increases the overall
metabolic rate of the subject, or an increase in free radical
flux.
3. The method of claim 1, wherein the induced intracellular
hyperthermia involve the induction of heat shock proteins.
4. The method of claim 1 further comprising administering an
anti-bacterial agent selected from the consisting of betalactam,
macrolide, tetracycline, aminoglycoside, peptide antibiotic,
sulfonamide, quinolone, nucleoside, oligosaccharide, polyene,
nitrofuran, and a combination thereof.
5. The method of claim 1 further comprising administering an
antiviral agent selected from the group consisting of amantadine,
rimantadine, arildone, ribaviran, acyclovir, abacavir, vidarabine,
9-1,3-dihydroxy-2-propoxy methylguanine, ganciclovir, enviroxime,
foscarnet, ampligen, podophyllotoxin, 2,3-dideoxytidine,
iododeoxyuridine, trifluorothymidine, dideoxyMosine, d4T, 3TC,
zidovudine, efavirenz, indinavir, saquinavir, ritonavir,
nelfinavir, amprenavir, and a combination thereof.
6. A method for inducing intracellular hyperthermia in a subject
comprising the step of administering to a subject having an
infestation of Sporothrix schenkii, Histoplasma, paracoccidiodes,
Aspergillus, Leishmania, malaria, acanthamoeba, or cestodes, an
amount of 2,4-dinitrophenol sufficient to induce whole body
intracellular hyperthermia in the subject, wherein the whole body
intracellular hyperthermia is sufficient to treat the Sporothrix
schenkii, Histoplasma, Paracoccidiodes, Aspergillus, Leishmania,
malaria, acanthamoeba or cestodes infestation in the subject.
7. The method of claim 6 further comprising administering an
antifungal agent selected from the group consisting of Amphotericin
B, Griseofulvin, Fluconazole, Intraconazole, 5 fluoro-cytosine,
Ketatoconazole and Miconazole.
8. The method of claim 6, wherein second medication is
administered, wherein the second medication increases the overall
metabolic rate of the subject, or causes an increase in free
radical flux.
9. The method of claim 6, wherein the induced intracellular
hyperthermia involve the induction of heat shock proteins.
Description
FIELD OF INVENTION
This invention relates to therapeutic pharmacological agents and
methods to chemically induce intracellular hyperthermia and/or free
radicals for the diagnosis and treatment of infections, malignancy
and other medical conditions. This invention further relates to a
process and composition for the diagnosis or killing of cancer
cells and inactivation of susceptible bacterial, parasitic, fungal,
and viral pathogens by chemically generating heat, free radicals
and hyperthermia-inducible immunogenic determinants. Such
pathogens, infected or transformed cells are inactivated or killed
without irreparable injury to non-transformed, uninfected, normal
cells. More specifically, this invention relates to the diagnosis
and treatment of cancer; treatment of AIDS; and, other diseases and
conditions using mitochondrial uncoupling agents, especially 2,4
dinitrophenol and, their conjugates, either alone or in combination
with other drugs, hormones, cytokines and radiation.
GENERAL BACKGROUND
Local heat, systemic hyperthermia and fever therapy have been
empirically used as effective treatments for malignant, infectious
and other diseases since antiquity. Therapeutic hyperthermia was
first documented in the Edwin Smith surgical papyrus in the 17th
century B.C. Coley's toxin extracts of Streptococcus erysipelatis
(group A streptococcus) and Bacillus prodigiosus (Serratia
marcescens) were used to induce fever for the treatment of patients
with advanced cancer. The Nobel Prize was awarded for using fever
therapy in the treatment of neurosyphilis with the injection of
malarial blood. As late as 1955, the Mayo Clinic advocated using
malariotherapy or heat therapy for cases of tertiary syphilis
"resistant to penicillin". Long term remissions in patients with
inoperable carcinomas that were treated with hot baths and local
heat applications have also been reported. Published observations
on the disappearance of malignancies such as a soft tissue sarcoma
in a patient experiencing high fever due to erysipelas and tumor
lysis of Burkitt's lymphomas following malignant hyperthermia
during surgical anesthesia are known. A comprehensive historical
review on anecdotal observations and intuitive rational for the
empirical use of therapeutic hyperthermia has been published by
Myer, J. L.
The temperature of a body can be intentionally increased either by
pyrogens to produce fever (fever therapy) or, by the induction of
hyperthermia (therapeutic hyperthermia). Fever raises body
temperature by elevating the thermoregulatory "set point" located
in the preoptic region of the anterior hypothalamus. In so doing,
the body physiologically works to maintain the higher temperature
setting. The elevated core body temperature increased by fever may
or may not be raised above the higher set point value. In contrast,
induced hyperthermia always raises the body temperature above the
hypothalamic thermoregulatory set point and the physiologically
intact body attempts to lower it's core temperature back to the set
point baseline.
Renewed clinical interest in hyperthermia has occurred over the
past 35 years due to continued failure of standard therapies to
treat various forms of cancer and emerging infections. Except for a
few exceedingly rare forms of cancer like childhood leukemias and
testicular cancer or immune responsive infections, chemotherapy,
radiation or drug therapy often do very little except briefly
extend survival. One of the major obstacles to "cure" disseminated
cancer and infections has been the innate or acquired resistance of
tumor cells and emerging microbes to antibiotics, drugs and
treatments given in tolerable doses. Escalation of treatments, or
use of multiple drugs to overcome resistance is invariably
prevented by concomitant toxicities or development of multi-drug
resistance. Further, in contrast to drugs, which represent a single
molecular species that biochemically interact with specific enzymes
or receptors of viruses, prokaryotes and eukaryotes, the action of
hyperthermia is biophysical and global. Hyperthermia has no
specific heat receptors. Therefore, the possibility of a point
mutation causing a functional change in a receptor and conferring
resistance to hyperthermia is unlikely, and would be equivalent to
the development of resistance to the in vitro process of
Pasteurization. Among pathogenic bacteria, it has been reported
that only one variant in 1.times.10.sup.6 cells of an original
population is resistant to hyperthermia.
Hyperthermia has been used alone or in conjunction with radiation
and chemotherapy in the treatment of a variety of malignancies.
Overgaard et al., reported that a combination of heat and radiation
results in complete control of twice as many melanoma lesions
compared to radiation alone. Maeda, M., Watanabe, N. et al.,
published in Gastroenterologia Japonica, that hyperthermia with
tumor necrosis factor resulted in successful treatment of
hepatocellular carcinoma. Prospective randomized studies of
hyperthermia combined with chemoradiotherapy for esophageal
carcinoma demonstrated the cumulative three year survival rates to
be more than doubled with the addition of hyperthermia to
chemoradiotherapy. Combination chemotherapy with hyperthermia in
metastatic breast cancer refractory to common therapies, i.e.,
failed prior hormonal therapy and chemotherapy, resulted in 39%
complete remissions and 23% partial remissions: relief of bone pain
was striking. Fujimoto, S., Takahashi, M. et al., demonstrated that
the 5 year survival rate of patients with peritoneal carcinomatosis
from gastric carcinoma treated with intraperitoneal hyperthermic
chemoperfusion was 41.6%, whereas the 50% survival duration of the
group that did not receive intraperitoneal hyperthermia was 110
days. Preoperative hyperthermia with chemotherapy and radiation is
also known to improve long-term results in patients with carcinoma
of the rectum, especially those with advanced disease. It is
clinically known that regional, i.e., limb, hyperthermic perfusions
with chemotherapy is useful for the treatment of melanoma.
Combination therapy with hyperthermia and radiation has been
successful in the treatment of non-Hodgkins lymphomas. More
recently, a survival benefit of hyperthermia was shown in a
prospective randomized trial for patients with glioblastoma
multiforme undergoing radiotherapy. However, rigorous clinical
prospective randomized trials with hyperthermia alone or, in
combination with agents outside its use with radiation therapy have
not been performed.
The scientific rationale for therapeutic hyperthermia in cancer
therapy rests on known data from pre-clinical, in vitro and animal
studies. Tumor cells in tissue culture have been demonstrated to be
directly more sensitive to heat as compared to their non-malignant
counterparts. Cells undergoing mitosis, synthesizing DNA in the
`S-phase`, are especially more sensitive to hyperthermia. Human
leukemic progenitor cells have been shown to be selectively killed
by hyperthermia and, such in vitro use has been shown to purge bone
marrow of residual tumor cells before autologous bone marrow
transplantation. Microcalorimetric measurements confirm that
tumorous tissues produce more heat and are "hotter" than their
non-tumorous counterparts. As a consequence, they are less able to
tolerate additional heat loads.
Tumor cells are also killed by heat indirectly. Tumor angiogenesis
is inhibited by heat. Hyperthermia causes tumors to have increased
heat retention with increased cytoxicity due to tumor
neovasculature lacking smooth muscle and vessel wall precursors
needed for cooling by vasodilation. Increased hypoxia, acidity, Fos
gene death signaling, decreased nutrient supply and enhanced
immunologic cytotoxicity have also been reported to be caused by
hyperthermia and contribute to enhanced tumor cell death. Further,
the combination of hyperthermia with chemotherapy and/or radiation
has been shown to be supraadditive or synergistic on killing of
tumors. Human gastric carcinoma cells have been shown to be
selectively killed by a combination of cisplatin, tumor necrosis
factor and hyperthermia: a 40% increase in cisplatin DNA damage was
noted in the presence of the three agent combination over cisplatin
alone or either dual combination. Numerous animal studies,
including the initial publication by Crile, show that neoplasms
transplanted into mice regress when treated with hyperthermia
without irreparable damage to adjacent tissues.
Body temperature is a critical factor in determining host
susceptibility, location of lesions, and the natural history of
many infectious diseases. Temperature has direct effects on the
growth of all microorganisms, including those that are pathogenic.
Almost all of the bacteria that cause disease in humans grow
optimally within the range of 33-41.degree. C. and, their
temperature growth characteristics are not easily altered in vitro.
By example, the lesions of Hansen's disease (leprosy) caused by
Mycobacterium leprae, characteristically grow and destroy the most
acral, coolest parts of the body such as fingers, toes, external
ear, the air-stream cooled nasal alae and larynx. Leprosy organisms
proliferate and follow the coolest temperature gradients in the
body, 25-33.degree. C. In animals, the leprae organisms can only be
grown in the armadillo or foot pads of mice where the in situ
lesion temperatures are 27-30.degree. C. Spontaneous improvement in
leprosy lesions have been reported in patients following febrile
illness. Fever therapy, hot baths and local heat therapy were
formerly utilized in treating this disease. Hyperthermia is also
known to destroy Treponema pallidum, the causative agent of
syphilis, by heating five hours at 39.degree. C., three hours at
40.degree. C., two hours at 41.degree. C. or one hour at
41.5.degree. C. The spirochetes responsible for yaws, bejel, pinta
and Lyme disease show similar temperature sensitivity.
Other bacteria that predominately cause lesions at cool sites and
are susceptible to heat inactivation include, Neisseria gonorrhea,
Hemophillus ducrei (chancroid), Mycobacterium ulcerans,
Mycobacterium marinum ("swimming pool" granuloma), Diphtheria, etc.
Further, hyperthermia has been reported to be synergistic with
antibiotic and chemotherapy in the treatment of various bacterial
diseases. Elevated body temperature potentiates the effect of
penicillin on staphylococci and syphilis. Hyperthermia makes
sulfadiazene bactericidal for streptococci. Moreover, recent
controlled studies show that when antipyretics are used in animals
with severe experimentally induced infections, there is increased
mortality. Nonetheless, systemic hyperthermia has generally been
abandoned as a treatment for bacterial infections with the advent
of antibiotics.
Hyperthermia has remained an effective treatment for many fungal
infections. Superficial dermatophytosis flourish in cooler regions
of the body and heat treatment is oftentimes the only viable
therapy for their chronic granulomatus lesions. By example,
Sporothrix schenkii, the causative agent of sporotrichosis, has a
temperature growth optimum well below 37.degree. C. and is
successfully eliminated by local hyperthermia. Similarly, patients
with pseudallescheriosis unresponsive to antifungal antibiotics are
healed with hyperthermic treatments. In Japan, pocket warmers, hot
water and infrared heating remain current and effective treatments
for various fungal infections. Systemic hyperthermia, utilizing a
Liebel-Flarsheim (Kettering) Hypertherm Fever Cabinet, dramatically
treated a case of disseminated sporotrichosis with recurrent
iridocyclitis, repeated post-treatment cultures from the patient
remained negative.
The role of hyperthermia in modulating the clinical course of other
fungal infections, including histoplasmosis, North American
blastomycosis, chromomycosis, cryptococcosis,
paracoccidioidomycosis, Lobos' disease and candidiasis has been
described. Fungi, such as Nocardia, Actinomyces and Aspergillus
also proliferate in cooler regions of the body causing mandible
(lumpy jaw) and foot lesions (Madura foot) respectively. In vitro
heat sensitivity data for many of the above and other pathogenic
fungi have been reported by Mackinnon et al., Silva and others.
The effect of temperature and hyperthermia on the pathogenesis of
parasitic disease is also well known. Leishmaniasis, a wide spread
parasitic disease transmitted by the bite of a sandfly, clinically
infects 12 million people worldwide. The cutaneous and
mucocutaneous lesions, i.e., Oriental sore, Baghdad boil, Delhi
boil, Chiclero's ulcer and espundia, are often very destructive and
permanently disfiguring. Hyperthermia with moist heat of 39.degree.
to 41.degree. C. applied for 20 hours over several days has proven
to be an effective treatment. In vitro, human macrophages infected
with Leishmania mexicana are completely destroyed by heating at
39.degree. C. for 3 days. All muco-cutaneous Leishmania strains,
regardless of subspecies, demonstrate a growth optimum of
35.degree. C. with only the L. tropica and L. donovani strains
surviving temperatures of 39.degree. C. Clinical observations have
shown that hyperthermic treatment of one Leishmania lesion often
invokes an immune response and results in the healing of other
lesions over a 5-6 week period. The effect of hyperthermia on other
parasites, including Trypanosoma cruzi, malaria, microfilaria,
acanthamoeba, trematodes and cestodes has been published.
Increased body temperature is also recognized as a major factor in
recovery from viral infections. Many viruses multiply better at
temperatures below 37.degree. C. and their multiplication is
inhibited or stopped if the body temperatures exceeds 39.degree. C.
In vitro Rhinovirus replication, for example, falls off by 10.sup.6
log units with an upward temperature shift of 2.degree. C.
(37.degree. to 39.degree. C.). Herpes virus replication, as well as
the intracellular and extracellular herpes virus concentration,
markedly decrease when the incubation temperature is elevated to
40.degree. C. Varicella virus production in human fibroblastic cell
culture is optimal at 37.degree. C. and ceases at 39.degree. C.
Beneficial effects of hyperthermia on the outcome of viral disease
in laboratory animals infected with myxomatosis,
encephalomyocarditis, herpes, gastroenteritis, rabies and the
common cold in man have been documented. Influenza and viruses
causing upper respiratory infections, such as the common cold,
thrive in a cool body milieu of 30.degree.-35.degree. C.
Temperature gradients in this range exist in the fall and winter
within the oral, nasal, tracheal and laryngeal mucosa and lead to
flu and influenza epidemics. Live respiratory-virus vaccines for
influenza have been developed by use of heat-sensitive mutants that
cannot reduplicate or cause clinical disease at
36.degree.-37.degree. C. It is known that even as little as a
0.5.degree. C. difference in the ceiling replication temperature of
a virus can have a dramatic effect on virulence and
pathogenicity.
Other animal viruses such as Newcastle disease in chickens, rabbit
papilloma, feline leukemia, rabbitpox, hoof-and-mouth disease in
cattle, hand, foot, and mouth disease, human plantar warts, and the
"grease" of horses, due to horsepox involvement of the colder acral
extremities above the fetlocks, are known to be very sensitive to
inhibition by heat. Heat treatment of cells infected with human
immunodeficiency virus (HIV-1) at 39.degree. C. for 2 days has been
documented to significantly decrease viral production and reduce
reverse transcriptase enzyme marker activity 30 fold. In vitro
hyperthermia of 42.0.degree. C. for 1 hour, 4 days apart
selectively lowers HIV RNA loads in chronic (latent) infected T
lymphocytes. Hyperthermia of 42.degree. C. for 3 hours combined
with tumor necrosis factor has been published to selectively kill
all acute and chronically infected HIV cells in tissue culture.
Use of whole body hyperthermia has been reported to cause
regression of Kaposis' sarcoma, clear oral candidiasis, eliminate
hepatitis C, cause remission of Varicella-zoster, increase weight
gain and improve CD4 lymphocytes counts in patients with acquired
immunodeficiency syndrome (AIDS). Dramatic improvement with
hyperthermia therapy has been documented in a patient infected with
a debilitating Verruca vulgaris and HIV. The FDA has approved
clinical trials involving hyperthermia for the treatment of AIDS
with a patented extracorporeal blood heating machine to induce
whole body hyperthermia. The FDA has recently expanded the
extracorporeal heating machine trials to permit treatment of 40 HIV
infected patients.
Hyperthermia can augment cytotoxicity and reverse drug resistance
to many chemotherapeutic agents. Moreover, hyperthermia has also
been shown to enhance the delivery of many novel cancer therapeutic
agents, i.e., monoclonal antibodies to neoplasms with resultant
improvement in antitumor effect; enhance the delivery of gene
therapy with use of viral vectors; and, augment drug delivery and
antitumor effects when using drug containing liposomes. In addition
to increasing the rate of extravasation of liposomes from the
vascular compartment by a factor of 40-50, hyperthermia can also be
used to selectively release chemotherapeutic agents from liposomes
designed to be thermosensitive. Thermosensitive liposomes are small
vesicles composed of lipid phosphatidylcholine moieties constructed
to contain and transport a variety of drugs. The liposomes are
designed to remain stable in the blood and tissues at physiologic
temperatures. When passing through an area of heated tissue
however, they dissolve and effectively release their encapsulated
contents. Thermosensitive liposomes are used to entrap and carry
drugs whose systemic toxicity is desired to be limited to a
particular heated tumor, organ or tissue. Examples of drugs that
have been encapsulated into liposomes include methotrexate,
doxorubicin, amphotericin B, cisplatin and others. Liposomes can be
designed so as to release their contents at pre-determined
temperatures.
Hyperthermia has also been an effective solution for the treatment
of a variety of heat labile toxin or poisonous envenomations. For
example, an easy treatment for Scorpaenidae and Siganidae
envenomation is the local application of heat. The major poisonous
component of this and many other venoms from lionfish, weever fish,
bullrout, sculpin, surgeon fish, scorpion fish, stonefish,
butterfly cod, etc., is a very heat labile, non-dialyzable protein.
As opposed to the nuances of using specific anti-venom, immersing
the envenomated area or patient in hot water, or applying other
forms of hyperthermia, is a simple and prompt treatment.
Standard clinical methods of inducing hyperthermia are dependent on
the deposition of exogenous heat to that normally produced by the
metabolism. All current deliberate and controlled methods of
heating require an external source of energy. Non-surgical methods
of heating include: hot air, ultrasound, microwaves, paraffin wax
baths, hot water blankets, radiant heat devices, high temperature
hydrotherapy and combinations thereof. Invasive means of inducing
hyperthermia include surgical insertion of various heating devices,
infusion of heated solutions into the peritoneal cavity through
catheters or heating the blood extracorporeally through a heat
exchanger. The later method, developed by Parks et al., involves
the surgical placement of a femoral arterio-venous shunt for the
removal, heating and replacement of blood to induce whole body
hyperthermia. A more recent experimental improvement on this method
has been the induction of whole body hyperthermia with veno-venous
shunt perfusions. Several machines have been patented for
extracorporeal heating of blood to induce hyperthermia (see U.S.
Pat. Nos. 5,391,142 and 5,674,190).
Endogenous heating by creating fevers induced with toxins, pyrogens
and microorganisms have been used in the past and have recently
been re-attempted. Heimlich has been reported to use Malaria
therapy for the treatment of Lyme disease, AIDS and malignancy.
Pontiggia et al, treated AIDS patients by combining fever, induced
by parenteral injections of a streptococcal lysate preparations,
with hyperthermia generated by an infrared heating bed.
Another way that the prior art has dealt with inducing hyperthermia
has been by introducing micron size magnetic particles and
subjecting them to either magnetic fields or hyperbaric oxygen (see
U.S. Pat. No. 4,569,836). This method was designed for the
treatment of cancer based on the belief that cancer cells would
engulf the particles and concentrate them intracellularly. A
magnetic field would then be applied to heat the particles and
generate lethal hyperthermia within the cancer cells. A
modification of this technology is the use of magnetic cationic
liposomes to induce intracellular hyperthermia. This technology was
based on the observation that glioma cells have a greater affinity
for positively charged rather than `neutral` magnetic liposomes. A
more recent variation on this science has been developed in Germany
using `targeted` magnetoliposomes. This methodology has been
developed in an attempt to treat AIDS by using magnetic
nanoparticles coupled to either CD4 lymphocyte or anti-gp120 HIV
antibodies. The magnetic nanoparticles are intended to selectively
bind to either the HIV protein envelope or the HIV infected cells
and then be heated by external high-frequency alternating magnetic
fields.
Whether invasive or non-invasive, all current methods of inducing
hyperthermia depend on an external energy source and cannot safely
deliver adequate power to result in therapeutic heating. Delivery
of heat to obtain the actual desired temperature to deep target
tissues has not been possible because of the actual physics
involved in the thermodynamic, conductive transfer of heat from the
outside into the cell. Heating tissues deeper than five centimeters
below the skin with microwave, radio frequency or ultrasound
devices is difficult because energy absorption is not uniform or
focused. Radiant heat, hot water, molten wax and other methods
cause excessive heating of subcutaneous fat which acts as a barrier
to body heat gain. Common adverse effects of such external heating
methods include surface skin burns, blistering, ulcerations,
secondary opportunistic infections and pain. Additionally, many
tumors have high blood flow cooling which nullifies any potential
therapeutic gain achievable through the use of such extracellular,
systemic hyperthermia devices. Also, insufficient heating power
prolongs the induction time required to reach the actual
therapeutic temperature. This promotes resistance to heat treatment
through the development of the heat shock response and
thermotolerance.
High frequency electromagnetic devices used to heat intracellular
magnetic particles invariably induce eddy currents within the body
making it difficult to provide uniform, controlled and safe heating
without toxic effects to normal cells. Further, not all tumors
possess characteristics that cause them to selectively take up
magnetic particles or have an affinity for positively charged
magnetic liposomes. Also, magnetic cationic liposome particles are
subject to various neutralizing interactions with anions, giving
them a short charged half-life. Moreover, the complexity of using
specific anti-HIV antibodies bound to electromagnetic particles
also assumes a non-mutating HIV genome with stable antigenic
determinants. To the contrary, a high mutation rate in the HIV
genome and it's protein antigenic determinants is known to exist
and is the main obstacle to the development of an effective
vaccine. Such treatments therefore, do not selectively heat
transformed cells without heating and injuring normal cells.
Extracorporeal blood heating methods require surgery and
anesthesia. Further, as with all external heating methods,
temperature variances and toxic conductive thermogradients from the
point of initial heating to the target tissue cannot be avoided. By
example, bone marrow temperatures are consistently known to be
1.degree.-2.degree. C. below the average body core temperature
achieved by extracorporeal blood hyperthermia. This is a major
problem in systemic hyperthermic therapy since the marrow is a
common repository of metastatic cancer cells and infectious
microorganisms. Therapeutic bone marrow temperatures are not
achievable due to the fact that the intermediate tissues between
the blood and the marrow create a temperature gradient cooling the
blood before it reaches the bone marrow. Since efficacy and
toxicity of hyperthermia depend on both the actual temperature and
duration of heating, delivering the desired
temperature-and-duration of heating (thermal dose) to the bone
marrow would require the blood and intermediate tissues to be
heated beyond that which is safe for normal, healthy cells. A
multicentre European trial documented that only 14% of all
protocols achieve required target temperatures. Further, current
extracorporeal heating methodology and equipment is labor
intensive, time-consuming and expensive.
Use of fever inducing agents such as live microorganisms, pyrogens
and toxin lysates is clinically uncontrollable, unpredictable or
insufficient as to both the degree and duration of temperature
increase.
Further reasons why hyperthermia has not yet become more widely
accepted as a mode of therapy is because current heating machines
are not compatible with noninvasive temperature measurement
technology. Measurement of the actual temperatures reached in
target tissues is critical for heating efficacy, i.e., determining
the thermal dose. Recently, noninvasive thermometry with Magnetic
Resonance Imaging (MRI), ultrasound backscatter, electrical
impedance, electromagnetic adaptive feedback and advanced,
high-precision pixel infrared temperature imaging have been
developed. To use MRI or other equipment to monitor real time
hyperthermia however, it is necessary to combine a hyperthermia
device with an MRI unit. This has proven to be difficult and costly
since each device is functionally disturbed, if not damaged, by the
presence of the other.
The exact molecular and cellular mechanism by which heat kills or
inactivates tumor cells and microorganisms is unknown. Heat is an
entropic agent and acts globally on every molecule constituting the
cell. Heating is known to cause conformational changes in proteins,
denature enzymes and affect cell membrane fluidity. By example,
herpes simplex virus (type 1) thymidine kinase has a shortened
half-life at 40.degree. C. of only 30 minutes. The transforming
gene product-enzyme of Rous sarcoma virus (protein phosphatase), a
critical protein for cellular regulation, is totally inactivated in
30 minutes at 41.degree. C. Hyperthermia is known to increase the
formation of oxygen free radicals, including superoxide, hydroxyl,
hydroperoxyl, hydrogen peroxide and lipid peroxides. These reactive
oxygen species react indiscriminately and oxidize many organic
molecules causing DNA damage, protein denaturation, lipid
peroxidation and other destructive chain reactions. Acid
microenvironments, known to exist in tumors and microorganisms with
high rates of glycolysis (Embden-Meyerhof Pathway) and lactic acid
production, favor protonation of the superoxide radical to form the
highly reactive and toxic hydroperoxyl radical. Thus, thermal
sensitivity of many tumors increases with decreasing intracellular
pH. As compared to normal cells, many malignant and virally
transformed cells have a reduced total functional capacity to
withstand the increase flux of oxygen free radicals produced by
hyperthermia.
On the intracellular level, moderate heating is known to activate
phospholipase A.sub.2, which increases the formation of
pro-inflammatory mediators such as the leukotrienes, prostaglandins
and eicosanoids. Heat also increases release of intracellular
calcium through the stimulation of phospholipase C. Calcium cycling
across the mitochondrial membrane appears critical to the increased
production of oxygen free radicals. Increased intracellular calcium
also inhibits the mitochondrial, anti-apoptotic Bcl-2 protein and
induces the production of heat shock proteins, mediating
thermotolerance. Heat injury to the intracellular tubulin network,
lysosomes, Golgi bodies, mitochondria, and control of RNA splicing
are some of the many known subcellular systems affected by heat.
While the initial primary event leading to cell death by
hyperthermia is unknown, a decrease in mitochondrial membrane
potential followed by uncoupling of oxidative phosphorylation and
generation of reactive oxygen species on the uncoupled respiratory
chain are the first biochemical alterations detectable in cells
irreversibly committed to apoptosis. The cytotoxic effect of
hyperthermia is thus believed to be caused by numerous changes and
complex damage to multiple vital cell functions. Those biochemicals
altered by heat and essential to the function or viability of the
cell are the pivotal targets of therapeutic heating.
The mode of hyperthermic cell injury is dependent on the severity
of the heat stress, temperature and duration of heating. Moderate
heating of 39.degree.-42.degree. C. is used therapeutically and is
known to promote programmed cell death through apoptosis, an active
process of selectively eliminating heat sensitive cells without
inflammation, bystander-cell death or subsequent tissue fibrosis.
Malignant and other transformed cells undergo apoptosis by
suppression or activation of one or more genes such as bcl-2,
c-myc, p53, TRPM-2, RP-2, RP-8, raf, abl, APO-11FAS, ced-3, ced-4,
ced-9, etc. Drugs (methotrexate, cisplatin, colchicine, etc.),
hormones (glucocorticoids), cytokines (tumor necrosis
factor-alpha), radiation (free radicals) and hyperthermia can all
initiate apoptosis. Increasing the temperature or duration of
heating, or both, leads to cell death via necrosis. This physical
process of indiscriminate cell killing is associated with
inflammation and causes significant injury to normal, healthy
cells.
For purposes of systemic hyperthermia, apoptosis of target cells is
the therapy of choice. In the clinical setting it must be
controlled under conditions of moderate heating so as to
selectively differentiate and eliminate target cells with minimum
toxicity to normal cells. Such controlled conductive heating by
external technologies is inherently not possible. The thermal
physical and thermophysiologic properties of cells vary and are
dependent on their thermal conductivity, specific heat, density and
blood perfusion among the various organs and tissues. Based on
these properties, the actual temperatures at some of these sites
are often `partitioned`, independent of one another and do not
represent the monitored, mean "core" temperature achieved during
therapy. Additionally, it is well recognized that it is the actual
intracellular temperature increase, with it's associated internal
physical and chemical changes, that is critical to the successful
use of hyperthermia in exploiting the fundamental biochemical
differences between normal and heat susceptible cells.
Unfortunately, the initial cellular targets of all extracorporeal
heating methods are the cell membrane and it's integrated proteins.
The cell's internal contents, including mitochondria,
compartmentalized enzymes, other organelles and any intracellular
pathogens, etc., are progressively heated in sequence by thermal
conduction from the outside-in. Thus, to sufficiently heat the
interior of the cell, the external temperature must overcome the
cellular and mitochondrial membranes, each composed of a lipid
bilayer that acts as an effective thermal barrier.
By necessity, therefore, prior art heating methods require high
external temperatures to establish a sufficient gradient to
overcome the nonisotropic and non-homogeneous conductive heat loss
between internal tissues and the insulating barrier of the cellular
and mitochondrial membranes. For example, the Organetics PSI.RTM.
(perfusion system (now First Circle Medical Inc.) device has to
heat blood externally to 480 C (118.40 F) before returning it
directly into the vascular system of the patient. Other
extracorporeal circuit perfusion devices need to achieve ex vivo
temperatures of 490 C (120.20 F.). Animal studies require
temperatures of 540 (129.10 F) during the induction phase to
achieve adequate target tissue temperatures. Safety in such prior
art is therefore limited by the incipient destruction of
surrounding tissues at the sites of the high temperature phases of
heating. When lesser temperatures are attempted, effectiveness is
compromised by either inadequate temperatures or duration of
beating or development of thermotolerance. As a result, only
regional hyperthermia has been widely used clinically and only in
combination with more traditional techniques such as radiation and
chemotherapy. Presently, none of the known heating technologies
provide clinically safe and effective hyperthermia to treat
systemic or disseminated disease. In order for systemic
hyperthermia to become more widely used clinically, current heating
methods must also overcome the use of labor intensive, complex
equipment including invasive extracorporeal infusion and it's
related toxicity problems to interposed tissues. Further, new
hyperthermic technology must be compatible with noninvasive, real
time thermometry.
The present invention avoids the problems of heat toxicity,
inadequate target tissue heating, excessive cost, surgery,
anesthesia and incompatibility with noninvasive temperature
measuring devices: problems that are inherent to all therapeutic
methods that deliver heat extracellularly, from the outside-in.
This invention is an intracellular, therefore, an intracorporeal
heating system which has additional distinct advantages. First, the
human body is biochemically and physiologically designed to
tolerate higher temperatures when heated from the inside-out as
opposed from the outside-in. By example, in comparison to
extracorporeal heating, which can safely generate a maximum body
core temperature of 42.degree. C. (107.6.degree. F.),
intracorporeal hyperthermia caused by strenuous exercise induces
physiologic temperatures of up to 45.degree. C. (113.0.degree. F.)
in muscle and liver with body core temperatures of up to 44.degree.
C. (111.2.degree. F.). Exertional heat stroke patients have
survived rectal body temperatures as high as 46.5.degree. C.
(115.2.degree. F.) without any permanent clinical sequela. While
the critical maximum temperature humans can tolerate is unknown,
physiologic hyperthermic temperature induced under controlled
conditions with adequate hydration have not shown any permanent
untoward effects. Liver biopsies from subjects with such
temperatures have not shown any significant microscopic
abnormalities. Second, since heating with the present invention is
chemically induced from within the cell, the actual intracellular
therapeutic temperature will be higher than the measured core
temperatures. As a result, intracellular organelles, including
mitochondria, are heated at higher temperatures, undergo greater
uncoupling and generate an increased flux of reactive oxygen
species. Since oxygen free radicals, including superoxide, enhance
and probably mediate the effects of hyperthermia, an improved
therapeutic gain will be obtained at lower body core temperatures.
Further, it is known that for each 0.5 degree Celsius increase in
body temperature the metabolic rate and oxygen consumption increase
7%. Such an increase will assist heating the body in itself. Third,
safety and control of temperatures with the present invention is
far superior to that of exogenous methods. The body is naturally
designed to dissipate heat from the inside-out. This is evident
from the fact that a temperature gradient of
3.5.degree.-4.5.degree. C. exists between the visceral core and the
skin. This gradient represents the transfer of heat from regions of
high temperature to regions of low temperature, with ultimate heat
loss from the skin to the environment through conduction,
convection, radiation and sweat induced evaporation. The margin of
safety and control represented by the `feedback gain` of this
intact physiologic heat dissipating system is extremely high,
approximating 27-33. This rate of cooling can balance an influx of
heat in a naked human body in a dry room at about 120.degree. C.
(248.0.degree. F.). Thus, the human heat flow system permits the
body to rid itself of excess endogenous heat very quickly and
effectively. As a result, there is a wide margin of safety in case
the target temperature is exceeded. In contrast, exogenous heating
contravenes the natural physiologic flow of heat and its
dissipating mechanisms. The natural heat dissipating mechanisms are
overwhelmed and compromised. Control and safety over hyperthermia
induced by extracellular means is thus fragile, with little room
for error.
SUMMARY OF THE INVENTION
The present invention encompasses a composition and method using
mitochondrial uncoupling agents, especially DNP, DNP with free
radical producing drugs, DNP with liposomes, DNP conjugated to free
radical formers, and DNP with other therapeutic pharmaceutical
agents which are activated intracellularly by heat or reaction with
mitochondrial electrons or free radicals to cause release of active
medications for the treatment of cancer, HIV, other viruses,
parasites, bacteria, fungi and other diseases. While not being
bound by theory, it is submitted that the use of mitochondrial
uncoupling agents, to increase intracellular heat and free
radicals, as treatment for non-related cancers, viruses and other
pathogens presupposes that the mechanism of action is non-specific
for enzymes and receptors but is specific for interference with
cellular and pathogen viability and induction of programmed cell
death. The degree of intracellular heating, free radical formation,
whole body hyperthermia and release of active drug molecules is
controlled by the dose of DNP. Based on the quantity of oxygen
consumed, the dose of DNP is adjusted to achieve the desired degree
of hyperthermia. Safety and effectiveness is further controlled by
manipulating metabolic rates of target tissues, duration of
treatment and permissiveness of body cooling. In accordance with
the present invention, intracellular, mitochondrial heat is
generated by the use of DNP, other uncouplers, their conjugates,
either alone or in combination with other drugs for the treatment
of thermosensitive cancers such as non-Hodgkins lymphoma, prostate
carcinoma, glioblastoma multiforme, Kaposi's sarcoma, etc; bacteria
such as Borrelia burgdorferi, Mycobacterium leprae, Treponema
pallidum, etc.; viruses such as HIV, hepatitis C, herpes viruses,
papillomavirus, etc.; fungi such as Candida, Sporothrix schenkii,
Histoplasma, Paracoccidiodes, Aspergillus, etc.; and, parasites
such as Leishmania, malaria, acanthamoeba, cestodes, etc.
2,4-dinitrophenol was selected as the uncoupler of choice because
it can be used at relatively high concentrations, permitting
uniform distribution in organs and tissues. This invention also
encompasses the use of DNP to selectively augment energy metabolism
and heat production in inchoate malignant tumors for the purpose of
increasing sensitivity of diagnostic positron emission tomography,
temperature-sensitive magnetic resonance, and high-precision pixel
temperature infrared imaging in differentiating normal from
aberrant cell metabolisms. An additional object of the invention is
the use of DNP to increase transcription of heat shock proteins,
especially HSP 72, as a form of cellular pre-conditioning to
decrease post-angioplasty restenosis, increase successful outcome
of other surgeries, and facilitate antigen processing and
presentation of immunogenic determinants on infectious agents,
virally transformed cells and tumors so as to increase the natural
or biologically activated immunological response.
In accordance with another aspect of the present invention,
controlled thermogenesis with DNP is combined with other agents
used to treat infectious, malignancy and other diseases. Examples
of other agents include antifungal, antiviral, antibacterial,
antiparasitic and antineoplastic drugs. Such drugs, including
angiogenesis inhibitors and radiation have increased synergistic or
additive activity when combined with hyperthermia in the treatment
of cancer.
The method can be used for enhancing the sensitivity of positron
emission tomography, nuclear magnetic resonance spectroscopy and
infrared thermography in the diagnosis and monitoring of treatment
of various diseases, including cancer. Similarly, the method can be
used for enhancing the identification of unstable "hot" coronary
and carotid artery plaques predisposed to rupture or undergo
thrombosis. Such diagnostic and treatment monitoring methodology is
based on the fact that most tumors have higher metabolic rates and
generate more heat than normal tissues. Likewise, unstable
atherosclerotic plaques are presumed to rupture because they have a
dense infiltration of macrophages which have high metabolic rates
and generate excessive enzymes and heat, causing the plaque to
degrade and loosen. In both instances, controlled doses of DNP or
other uncouplers can further increase metabolic rates and heat
production to increase diagnostic sensitivity. Controlled heating
with DNP and fibrinolytic recombinant tissue-type plasminogen
activators can also be used therapeutically to accelerate
fibrinolysis of clotted arteries.
In another aspect of the invention, DNP is administered in
controlled and timed dosages to provide physiologic stress,
"chemical exercise", so as to induce synthesis of autologous heat
shock proteins (HSPs). Intracellular heat exposure associated with
autologous HSP induction has a significant cytoprotective effect
against ischemia and cellular trauma and acts as a form of cellular
thermal preconditioning in patients about to undergo surgery.
Induction of HSPs by DNP in patients some 8 to 24 hours prior to
angioplasty, coronary bypass surgery, organ transplantation and
other forms of high risk surgery, would provide for improved
clinical outcome with decreased post-angioplasty intimal thickening
or restenosis, increased myocardial protection from infarction,
improved musculocutaneous flap survival in plastic reconstruction
and reduced ischemia/reperfusion injury in organ transplantation
cases.
Another aspect of the invention provides for controlled dosages of
DNP to induce long duration (6 to 8 hour), mild whole body
hyperthermia (39.0 to 40.0.degree. C.) to afford maximum expression
of immunogenic HSPs or peptides associated with HSPs. The antigenic
properties of HSPs and HSP-peptide complexes, induced by DNP in
infectious agents, especially those located intracellularly, or on
tumors can be exploited to enhance the immune response. This aspect
of the present invention provides a process for modulating the
immune system of a patient with other therapies, comprising the
steps of: (1) increasing the expression of HSPs by the process
described above, and (2) administering humanized monoclonal or
polyclonal antibodies, or (3) administering recombinant cytokines,
lymphokines, interferons, etc., or (4) administering standard
anti-infectious or anti-neoplastic therapy.
Additional objects and advantages of the invention will be set
forth in part in the description of drawings that follows, and in
part, will be obvious from the description, or may be learned by
practice of the invention. The objects and advantages can be
realized and obtained by means of the uses and compositions
particularly pointed out in the detailed description of the
preferred embodiments and in the appended claims.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows features of glycolysis with formation of pyruvic acid
and release of energy as heat.
FIG. 2 depicts the conversion of pyruvic acid into acetyl CoA and
the 2 carbon fragments entering the TCA cycle.
FIG. 3 shows the transfer of electrons down the electron transport
chain during the process of oxidative phosphorylation
FIG. 4 shows oxidative phosphorylation as a coupling of two
distinct processes, oxidation of reducing equivalents and formation
of ATP. Both processes are "coupled" by an electro-chemical
membrane potential created by electrons passing down the electron
transport chain.
FIG. 5 shows the process of chemiosmosis. Electrons passing down
the electron transport chain create energy to pump H.sup.+ outside
the inner mitochondrial membrane. This process creates a
protonmotive force that causes formation of ATP by protons
re-entering the membrane through ATP-synthase.
FIG. 6 depicts the uncoupling of oxidative phosphorylation through
injury of the inner mitochondrial membrane. FIG. 6(a) shows how
oxidative phosphorylation is uncoupled by DNP in intact and
uninjured mitochondrial membranes.
FIG. 7 shows the initial formation of superoxide radicals by the
univalent reduction of oxygen in the electron transport chain. FIG.
7(a) depicts the formation of hydrogen peroxide and hydroxyl
radicals through the Haber-Weiss Reaction. FIG. 7(b) shows an
overview of mitochondrial oxygen utilization and free radical
formation.
FIG. 8 depicts the effects of heating on mitochondrial uncoupling
and correlation of uncoupling to superoxide free radical formation.
FIG. 8a shows the relationship between mitochondrial respiratory
control ratio and superoxide production. FIG. 8b shows the
relationship between mitochondrial upcoupling and superoxide free
radicals.
FIG. 9 depicts the increased formation of oxygen free radicals
after cessation of DNP uncoupling and normalization of oxygen
consumption.
FIG. 10 shows the global intracellular effects of DNP, including
the dominant foci of increased heat generation.
FIG. 11 shows the relative potencies of various uncouplers.
FIG. 11(a) shows the effect of body temperature on metabolic
rate.
FIG. 12 shows six of the Hottest organs in the human body and their
relative blood flow.
FIG. 13 shows the effect of successive doses of 2,4-DNP on oxygen
consumption.
FIG. 14 shows a typical DNP induced hyperthermia patient monitored
flow chart.
FIG. 15 shows a monitored patient flow chart after successive
infusions of DNP and glucagon for treatment of parasitic disease of
the liver.
FIG. 16 shows killing of chronically HIV infected HUT-78 cells with
varying concentrations of DNP.
FIG. 17 shows a patient flow chart after infusion of norepinephrine
and successive intravenous doses of DNP for treatment of HIV
disease. FIG. 17(a) depicts surrogate parameters relating to HIV
disease before and after DNP treatment.
FIG. 18 shows a monitored patient flow chart after successive
infusion of DNP for treatment of Lyme disease.
FIG. 19 shows a monitored patient flow chart using an alpha-1
adrenergic agonist with DNP to induce hyperthermia in a patient
with disseminated cancer.
FIG. 20 shows survival studies of tumor growth-regressed animals
treated with DNP and a thermosensitive liposome encapsulated
drug.
FIG. 21 shows the protective effects of DNP pretreatment on
arterial catheter balloon induced injury.
FIG. 22 shows the protective effects of DNP pretreatment on
survival after prolonged hepatic eschemic induced by Pringle's
maneuver.
FIG. 23 shows the improved effect of musculocutaneous flap skin
survival after DNP pretreatment.
FIG. 24 shows the effects of oral DNP on oxygen consumption prior
to a patient undergoing a PET scan.
FIG. 25 shows a monitored DNP flow chart with incremental increases
in oxygen consumption prior to a patient undergoing diagnostic
thermography.
FIG. 26 shows a monitored patient flow chart using dinitrophenol
and methylene blue for the treatment of prostate carcinoma.
FIG. 27 shows biochemical and clinical response of
androgen-independent prostatic carcinoma to dinitrophenol and
methylene blue treatment.
FIG. 28 shows a monitored patient flow chart using interferon-alpha
and dinitrophenol for the treatment of chronic hepatitis C
infection.
FIG. 29 shows the effects of dinitrophenol and interferon-alpha
treatment on liver enzymes and hepatitis C viral loads.
FIG. 30 shows an exemplary method of synthesis of novel
2,4-dinitrophenol conjugates and derivatives.
FIG. 31 shows synthesis of an expanded combinatorial library of
uncoupling agents.
DETAILED DESCRIPTION OF THE INVENTION
Electron transferring, transporting and energy converting elements
are ubiquitous and are necessary for life. All eukaryotic and
prokaryotic organisms depend on electron transferring and
transporting elements such as metal containing hemes and nonmetal
moieties such as flavins and adenine nucleotides. These biochemical
entities convert the energy stored in chemical bonds of foodstuffs
into cellular and organelle membrane potentials, high energy
containing molecules such as adenosine triphosphate (ATP),
creatinine phosphate, and other forms of chemical energy needed to
maintain the highly negative entropic state of life.
The most common form of biologic energy is adenosine triphosphate
(ATP). ATP is produced either anaerobically through the
Embden-Myerhoff Pathway (glycolysis) or through oxidative
phosphorylation. The latter, an oxygen dependent chemical energy
conversion process, is generally associated with the Tricarboxylic
Acid Cycle [(TCA), Krebs Cycle or Citric Acid Cycle]. The TCA cycle
links the products of glycolysis to a multi-enzyme coupled series
of electron carriers called an electron transport chain (ETS). The
electron transport chain is coupled to production of ATP. The
entire TCA cycle and oxidative phosphorylation process is located
in intracellular organelles known as mitochondria.
While release of energy from foodstuffs can come about through a
variety of biochemical means, the most important means by which
energy release is initiated is by splitting glucose into two
molecules of pyruvic acid. This occurs through the non-oxygen
dependent process of glycolysis in a series of ten chemical steps
depicted in FIG. 1. The overall efficiency of trapping energy in
the form of ATP through this anaerobic process is 43%. The
remaining released energy (57%) is discharged in the form of
heat.
Pyruvic acid molecules derived from glucose, as well as end
products of fat and protein breakdown, are transported into the
mitochondrial matrix where they are converted into 2 carbon
fragments of acetylcoenzyme A, FIG. 2. As depicted, these acetyl
fragments enter the TCA cycle were their hydrogen atoms are removed
and released as either hydrogen ions (H+) or combined with
nicotinamide and flavin adenine dinucleotides (NAD+ and FADH) to
produce large quantities of usable reducing equivalents (NADH and
FADH2). The carbon skeleton is converted to carbon dioxide (CO2)
which becomes dissolved in body fluids. Ultimately the dissolved
CO2 is transported to the lungs and expired from the body. As noted
in FIG. 2, the flux of reactants in the TCA cycle is always in the
same direction because NADH and FADH2 is constantly removed as
hydrogen is oxidized by the mitochondrial electron transport
chain.
It is the electron transport chain that provides approximately 90%
of the total ATP formed by glucose catabolism. During this process,
known as oxidative phosphorylation, hydrogen atoms that were
released during glycolysis, the TCA cycle, and converted to NADH
and FADH.sub.2, are oxidized by a series of enzymatic redox
complexes (electron transport chain) located in the inner
mitochondrial membrane, FIG. 3. Energy released in these steps is
captured by a chemiosmotic mechanism that is dependent on the
ultimate reduction of O.sub.2 to form H.sub.2O. As depicted in FIG.
4, oxidative phosphorylation is two distinct processes: (1)
oxidation of NADH and FADH.sub.2; and, (2) formation of ATP. Both
processes are interdependent or "coupled" by a high energy linked
proton (H.sup.+, pH) gradient and membrane potential across the
inner mitochondrial membrane provided by electrons as they pass
through the electron transport chain. Energy released by the
electrons pumps hydrogen ions (H.sup.+) from the inner matrix of
the mitochondrion into the outer inter-membrane space, FIG. 5. This
process is known as chemiosmosis and creates a high concentration
of H.sup.+ outside the inner mitochondrial membrane and a powerful
negative electrical potential in the inner matrix. This
transmembrane proton gradient (protonmotive force) causes hydrogen
ions to flow back into the mitochondrial matrix through an integral
membrane protein (ATP synthase) to form ATP from ADP and free ionic
phosphate. The efficiency of oxidative phosphorylation in capturing
energy as ATP is about 69%. The remaining (31%) liberated energy is
dissipated as heat. The overall efficiency of energy transfer to
ATP from glucose via glycolysis, the TCA cycle and oxidative
phosphorylation is 66% with about 34% of the energy being released
as heat.
Heat is continually produced by the body as a byproduct of
metabolism and eventually all energy expended by the body is
converted to heat. On a thermodynamic basis, total body heat
production is the algebraic sum of the enthalpy changes of all
biologic processes in the body. The pathways are irrelevant, even
though in the body oxidation involves numerous enzyme catalyzed
reactions taking place at 37.degree. C. Biochemically,
approximately 95% of all the oxygen (O.sub.2) consumed is used by
mitochondria to stoichiometrically couple oxygen reduction to ATP
and heat production via oxidative phosphorylation. The rate of
O.sub.2 consumption (VO.sub.2) can be measured by indirect
calorimetry and thus related to body heat production. Although this
method does not include anaerobic processes such as glycolysis,
indirect calorimetry is in close agreement with direct body heat
measurements and it is generally accepted that 1 liter of VO.sub.2
generates 4.825 Kcal (kilocalorie of energy), .sup.ths of which can
be detected as heat.
In human adults, increased VO.sub.2 and endogenous heat production
can occur via muscular (work or shivering) and/or chemical
[(cathecholamines, thyroid, etc.) non-shivering] thermogenesis.
Whereas muscular activity can increase heat production 4-10 fold,
non-shivering thermogenesis can only increase heat production by a
maximum of 15%. However, oxygen consumption and non-shivering
thermogenesis can dramatically increase when even mild injury to
the inner mitochondrial membrane occurs so that it is no longer
intact and protons leak or reenter the mitochondrion, uncoupled to
ATP synthesis. Heating, endotoxin, osmotic imbalance, etc., can
cause such injury, i.e., loss of coupling, with resulting
respiration and ATP metabolism proceeding independently and
maximally--respiration forward, phosphorylation in reverse. FIG. 6
compares normal coupled respiration and ATP formation to that which
occurs when there has been injury to the inner mitochondrial
membrane. The increased reduction of oxygen results in increased
heat production.
Additionally, certain chemicals, including biologicals, can
selectively increase the transport of protons across uninjured,
intact inner mitochondrial membranes and dramatically increase
VO.sub.2 and heat production. These compounds dissipate the
electrochemical-protonmotive transmembrane potential of
mitochondria and uncouple the electron transport chain from ATP
synthesis. FIG. 6(a) depicts one such uncoupling agent, DNP,
cycling protons across an intact mitochondrial membrane. DNP and
other uncouplers permit each of the two distinct processes involved
in oxidative phosphorylation to "unlink" and increase their rates
according to their own separate kinetic and thermodynamic signals,
FIG. 6(b). Uncouplers increase respiratory rates, electron
transport, VO.sub.2, heat production and increased utilization of
foodstuff substrates through glycolysis and the TCA cycle.
Controlled doses of an uncoupler will increase 0.sub.2 consumption
and heat production with minimal or no decrease in ATP levels
because of intracellular equilibrium shifts in creatinine
phosphate, oxidative phosphorylation reactants and increased
production of ATP through the anaerobic, glycolytic pathway. Excess
or toxic doses of virtually all uncouplers however, will produce
secondary untoward effects, including decreased respiration,
decreased heat production and eventual cellular death.
In addition to heat being a byproduct of oxidative phosphorylation,
reactive oxygen species are also continuously produced by the
mitochondrial electron transport chain. Free radicals of oxygen are
produced during aerobic oxidation as electrons are transported by
the electron carriers to ultimately reduce O.sub.2 to H.sub.2O. As
depicted in FIG. 7, superoxide (O.sub.2.sup.-) radicals are
generated by leaked electrons through the univalent reduction of
oxygen. FIG. 7(a) shows that superoxide dismutase then converts the
superoxide radical to hydrogen peroxide. Additional hydrogen
peroxide (H.sub.2O.sub.2) and hydroxyl (OH.) radicals are formed
through the Haber-Weiss Reaction, the hydroxyl radical being the
most reactive species, reacting with any biologic moiety instantly.
FIG. 7(b) depicts the overall scheme of oxygen metabolism and free
radical formation at the level of the mitochondrion.
As mitochondria become progressively heated, uncoupling occurs with
increased flux of oxygen free radicals. The effects of heat on
mitochondrial uncoupling and superoxide radical generation are
depicted in FIG. 8. A linear correlation of 0.98 (P<0.01) is
obtained for the relationship between percent uncoupling and
percent superoxide generation. Similar to exercise increased body
temperature and VO.sub.2, hyperthermia induced by uncoupling agents
appears to inhibit electron transport at the level of cytochrome c
in the redox chain. Normal rat liver, infused with DNP, increases
formation of reactive oxygen species threefold upon cessation of
uncoupling, FIG. 9.
Generally, uncouplers are agents that are hydrophobic ionophores
which bind protons and traverse biologic membranes to dissipate
transmembrane proton (pH) and membrane potential gradients
(.DELTA..PSI., Delta Psim). In so doing, uncouplers increase the
rate of metabolism (substrate utilization) in intact animals and
isolated tissues by increasing the rate of oxygen reduction through
increased availability of protons. 0.sub.2 consumption is increased
and remains rapid as long as the mitochondrial respiratory
(electron transport) chain attempts to overcome the effects of the
uncoupler to maintain a pH gradient. Energy is still used to pump
protons across the mitochondrial membrane, but the protons are
carried back across the membrane by the uncoupler as depicted in
FIG. 6(a). This creates a futile cycle and energy is released as
heat. This chemical heat releasing process is comparable to heating
that occurs when an electrical wire is "short circuited". Depending
on the degree of external body heat dissipation, body temperature
rises some 30 to 60 minutes after the increase in 0.sub.2
consumption. Onset of action is rapid after an intravenous
injection of an uncoupler. Depending on the intravenous dosage,
human oxygen consumption is increased in about 15-20 minutes and
the intracellular heat production is increased proportionately.
Metabolic rates as high as 10 times normal have been reported.
Persistent increases in the metabolic rate can continue as long as
12 to 36 hours because of the long hydrophobic half-life of
uncouplers in tissues. Temperature increases can be seen within 10
to 15 minutes in subjects whose heat dissipation mechanisms have
been compromised. Heretofore, hyperthermia induced by uncoupling
compounds has not been reported to have any therapeutic
application.
While there are three general classes of uncoupling agents, each
containing specific uncouplers of oxidative phosphorylation, the
present invention utilizes 2,4-dinitrophenol (DNP) as the preferred
embodiment. This is because DNP has been extensively studied. DNP
was commonly used in food dyes in the late 1800's and in the
munitions industry of World War I. Rapid increased respiration and
hyperthermia, up to 49.degree. C., was noted in man and animals
that were accidentally intoxicated. Such dramatic physiologic
effects by the dinitro-aromatic dyes, especially DNP, caused them
to be inextricably tied to early and later modern studies of
metabolism and bioenergetics. In the 1930's DNP was introduced into
clinical medicine for the purpose weight loss. It was, however,
sold as an over the counter secret nostrum and seriously misused.
Had its long half-life in tissues been recognized and physician
supervision implemented, it might have become an accepted drug. DNP
has been reported in countless, different enzyme, cellular and
metabolic studies. Review of such vast published studies have
documented DNP's very specific mechanism of action as a proton
ionophore, with all other effects a direct pharmacologic extension
thereof. DNP is not mutagenic by the Ames and modified Ames tests;
it has not been found to be carcinogenic or teratogenic; and, DNP
blood plasma levels can easily be determined. DNP can be used at
pharmacologic doses that achieve therapeutic concentrations in
tissues. Further, DNP is stable, inexpensive and commercially
available in reagent grade purity. It is understood however, that
other uncouplers and combinations of other uncouplers with other
drugs, hormones, cytokines and radiation can potentially be used
under appropriate clinical settings and dosages to induce
intracellular hyperthermia and promote additive or synergistic
effects.
FIG. 10 shows the overall intracellular mechanism of action of DNP
(and other uncouplers). Intracellular foci of increased heat and
oxygen free radical flux are highlighted. Circled numbers in the
figure indicate both direct and indirect effects of DNP: circled 1
and 2 effects shows that upon its intercalation into the inner
mitochondrial membrane, DNP shuttles H.sup.+ (hydrogen ions) across
the membrane [see FIG. 6(a)]--this short circuits (de-energizes)
the proton gradient established by the H.sup.+ pumping action of
the mitochondrial electron transport system (see FIG. 5). As a
consequence, the inner mitochondrial membrane potential is lowered
from -180 to -145 mV. Circled 3, 4, 5 and 6 effects shows that
normal oxygen consumption and flux of NADH and FADH.sub.2 (reducing
equivalents) through the electron transport system is coupled to
H.sup.+ re-entry via mitochondrial availability of ADP for
re-synthesis of ATP (see FIG. 4). By freely returning protons into
the mitochondrial matrix without concomitant dependency on ADP to
ATP reformation, DNP increases oxygen consumption proportionately
to the degree of uncoupling. The rate of oxygen consumption remains
linked however, to the flux of electrons provided by NADH and
FADH.sub.2 through the electron transport chain [see FIG. 6(a)].
NADH and FADH.sub.2 utilization (re-oxidation) is concomitantly
increased. Circled 7, 8, 9, and 10 effects show that oxygen use and
electron transfer proceed at increasing rates to accelerate proton
pumping against the added hydrogen ion load introduced by DNP. As a
result, NADH and FADH.sub.2 is continually depleted by re-oxidation
to NAD.sup.+ and FAD.sup.++. The high "oxidation pressure" of
NAD.sup.+ and FAD.sup.++ increases substrate oxidation and flux of
2 carbon segments through the tricarboxylic acid cycle (TCA).
Augmented acetyl-CoA consumption in turn is maintained by an
increased rate of glycolysis by depletion of pyruvate. If oxygen
delivery is inadequate, or the dose of DNP excessive, the
concentration of reduced NADH increases, pyruvate oxidation through
acetyl-CoA and the TCA cycle is inhibited and lactic acid will
accumulate. Lactate is also overproduced when cellular hypoxia is
not present per se but glycolysis exceeds pyruvate oxidation. Such
intracellular lactic acidosis exists in neoplastic cells, when
there is lack of insulin, when fructose is infused and in other
conditions or use of drugs which augment glycolysis and/or inhibit
the mitochondrial electron transport system. While it is understood
that the intracellular heat generated by DNP is the algebraic sum
of the enthalpy changes from all the metabolic processes within the
cell, effects circled as 11, 12 and 13 depict the most significant
intracellular foci of heat generated by DNP. Intracellular and
total body hyperthermia results when DNP releases energy at a rate
faster than it can be dissipated. Heat is generated mainly at the
inner mitochondrial membrane (electron transport system), the TCA
cycle and sites of cytoplasmic glycolysis. Initially DNP generates
heat at the inner mitochondrial membrane by discharging a portion
of the energy stored in its electrochemical gradient.
Operationally, such heat is from the "chemical short circuit"
created by DNP shuttling protons to the negative (matrix) side of
the polarized inner mitochondrial membrane [see FIG. 6(a)]. By
usurping controlled proton re-entry and energy capture as ATP from
availability of ADP through ATP-synthase, DNP causes NADH and
FADH.sub.2 (higher concentrations of NAD.sup.+ and FAD.sup.++)
reoxidation to occur at rates much higher than necessary for
oxidative phosphorylation. This causes an increased fall of
electrons through the electron transport chain with rapid reduction
of oxygen to water (see FIG. 3). The resultant energy is released
as heat within the mitochondrial membrane. The rate of heat
production from the TCA cycle is increased as it operates at a
higher flux to maintain depleting amounts of reduced NADH and
FADH.sub.2 used to reduce molecular oxygen. Flux of acetyl-CoA and
all metabolites through the TCA cycle (see FIG. 2) is increased by
activation of enzymes which sequentially degrade the hydrogen
containing two carbon fragments to CO.sub.2, NADH, FADH.sub.2 and
heat.
Glycolysis and its associated heat production in the cytoplasm is
also increased by DNP. Glycolytic activity is increased by reduced
concentration ratios of ATP to ADP, activating pyruvate
dehydrogenase and phosphofructokinase respectively (see FIG. 1).
These enzymes increase the rate of glucose catabolism to pyruvate
and its conversion to acetyl-CoA for entry into the TCA cycle.
Glycolysis is very "energy inefficient" in making up the energy
equilibrium shortfall created by DNP. Uncaptured energy from the
glycolytic exergonic reactions accelerated by DNP is released as
heat in the cytoplasm DNP stimulated anaerobic heat production
through glycolysis can oftentimes be greater than that produced by
the mitochondria. By example, many tumors and normal fibroblasts
treated with DNP increase heat production by 83%, with only a 36%
increase in oxygen consumption. Glycolysis is known to contribute
greater than 62% of the total heat produced by human lymphocytes.
Circled effect 14 shows that the mitochondrial electron transport
chain normally produces reactive oxygen species through the
univalent reduction of oxygen [see FIG. 7, 7(a) & 7(b)]. Under
physiologic conditions, 2 to 4% of mitochondrial oxygen is
converted to superoxide. DNP induced partial uncoupling and
mitochondrial heating increases reactive oxygen species production
manifold. Cytochrome oxidase and reductase is known to be inhibited
by heating of the electron transport system. As a result, heated
mitochondrial membranes produce increased amount of oxygen free
radicals when DNP induced uncoupling is stopped and oxygen
consumption is normalized (see FIG. 9). Reactive oxygen species act
in synergy with beat to alter proteins, induce membrane changes and
initiate apoptosis in susceptible cells. Circled effects 15 and 16
shows the effects of DNP on intracellular calcium homeostasis.
Normally calcium is stored in the mitochondrial matrix, being
pumped by the energized mitochondrial membrane. By DNP directly
de-energizing mitochondria, and indirectly inducing membrane
heating and prooxidant stress, inner mitochondrial membrane
permeability is non-specifically increased with calcium efflux and
cycling. This activates intramitochondrial dehydrogenases to
produce more reducing equivalents in the form of NADH and FADH2 to
match increased energy demands. Heat production is increased as a
byproduct from the augmented TCA cycle.
Other known uncouplers that are considered to be "classic", in the
same category and act as DNP include clofazimine, albendazole,
cambendazole, oxibendazole, triclabendazole (TCZ),
6-chloro-5-[2,3-dichlorophenoxyl]-2-methylthio-benzimidazole and
their sulfoxide and sulfone metabolites, thiobendazole, rafoxanide,
bithionol, niclosamide, eutypine, various lichen acids
(hydroxybenzoic acids) such as (+)usnic acid, vulpinic acid and
atranorin, 2',5-dichloro-3-t-butyl-4'-nitrosalicylanilide (S-13),
3,4',5-trichlorosalicylanilide (DCC), platanetin,
2-trifluoromethyl-4,5,6,7-tetrachlorobenzimidazole (TTFB), 1799,
AU-1421,
3,4,5,6,9,10-hexahydro-14,16-dihydroxy-3-methyl-1H-2-benzoxacyclotetradec-
in-1,7(8H)-dione (zearalenone),
N,N.sup.1-bis-(4-trifluoromethylphenyl)-urea, resorcylic acid
lactones and their derivatives,
3,5-di-t-butyl-hydroxybenzylidenemalononitrile (SF6847), 2,2-bis
(hexafluoroacetonyl)acetone, triphenyl boron, carbonylcyanide
4-trifluoromethoxyphenylhydrazone (FCCP), tributylamine (TBA),
carbonyl cyanide 3-chlorophenylhydrazone (CICCP),
1,3,6,8-tetranitrocarbazole, tetrachlorobenzotriazole,
4-iso-octyl-2,6-dinitrophenol (Octyl-DNP),
4-hydroxy-3,5-diidobenzonitrile, mitoguazone (methylglyoxal
bisguanylhydrazone), pentachlorophenol (PCP),
5-chloro-2-mercatobenzothiazole (BZT-SH), tribromoimidazole (TBI),
N-(3-trifluoromethylphenyl)-anthranilic acid (Flufenamic acid),
4-nitrophenol, 4,6-dinitrocresol, 4-isobutyl-2,6-dinitrophenol,
2-azido-4-nitrophenol, 5-nitrobenzotriazole,
5-chloro-4-nitrobenzotriazole, tetrachlorobenzotriazole,
methyl-o-phenylhydrazone, N-phenylanthranilic acid,
N-(3-nitrophenyl)anthranilic acid,
N-(2,3-dimethylphenyl)anthranilic acid, mefenamic acid, diflunisal,
flufenamix acid, N-(3-chlorophenyl)anthranilic acid, carbonyl
cyanide 4-trifluoromethoxyphenylhydrazone (FCCP), SR-4233
(Tirapazamine), atovaquone, carbonyl cyanide
4-(6'-methyl-2'-benzothiazyl)-phenylhydrazone (BT-CCP),
ellipticine, olivacine, ellipticinium, isoellipticine and related
isomers, methyl-0-phenylhydrazonocyanoacetic acid,
methyl-0-(3-chlorophenylhydrazono) cyanoacetic acid,
2-(3'-chlorophenylhydrazono)-3-oxobutyronitrile, thiosalicylic
acid,
2-(2',4-dinitrophenylhydrazono)-3-oxo-4,4-demethylvaleronitrile,
relanium, melipramine, and other diverse chemical entities
including unsaturated fatty acids (up to C.sub.14 optimum),
sulflaramid and its metabolite perfluorooctane sulfonamide (DESFA),
perfluorooctanoate, clofibrate, Wy-14, 643, ciprofibrate, and
fluoroalcohols. Additional unnamed classic uncouplers can include
any analog which generally has a weakly acidic, removable proton
and an electron withdrawing, lipophilic molecular body that is
capable of charge delocalization. Hydrophobicity and capacity to
exchange proton equivalents are integral features of classic DNP
types of uncouplers.
A second class of uncouplers are ionophorous antibiotics. These
molecules uncouple oxidative phosphorylation by inducing cation or
anion influx across the mitochondrial membranes and diffusing back
in a protonated form. As a result, chemical futile cycling ensues
to reestablish the initial membrane potential. Liberated energy is
dissipated as heat. Examples of ionophores that shuttle potassium
ions (K.sup.+) across membranes includes the antibiotics
gramicidin, nigericin, tyrothricin, tyrocidin, and valinomycin.
Nystatin shuttle sodium ions. The calcium ionophore, compound
A23187, is a lipid soluble ionophore which mediates the
electroneutral exchange of divalent cations for protons.
Alamethicins, harzianin HA V, saturnisporin SA IV, zervamicins,
magainin, cecropins, melittin, hypelcins, suzukacillins, monensins,
trichotoxins, antiamoebins, crystal violet, cyanine dyes, cadmium
ion, trichosporin-B and their derivatives are examples of
uncoupling ionophores that depend on shuttling inorganic phosphate
(P0.sub.4.sup.=) across the mitochondrial membrane.
A third class of uncouplers is a group of heterogeneous compounds
that dissipate the proton gradient by attaching or interacting with
specific proteins in the inner mitchondrial membrane. Examples of
such compounds include desaspidin, ionized calcium (Ca.sup.++),
uncoupling proteins such as UCPI-1, UCP-2, UCP-3, PUMP (Plant
Uncoupling Mitochondrial Protein) histones, polylysines, and
A206668-a protein antibiotic that ties up phosphoryl-transfer
proteins. Examples and a potency comparison of a few uncouplers are
depicted in FIG. 11.
Various conjugates, adducts, analogs and derivatives of the above
mentioned agents can be formulated and synthesized to enhance
intracellular uncoupling and heat production. Further, various
covalent compounds of uncouplers may be synthesized as prodrugs,
which upon, redox or reaction with free radicals within the cell
will become activated to induce uncoupling, heat production and
free radical cycling. Such derivatives and formulations may be
desirable in the treatment of many tumors with higher mitochondrial
membrane potentials and increased total bioreductive capacity.
Uncoupling-free radical prodrug compounds may thus exert greater
selective killing of transformed cells by undergoing a higher flux
of reduction or electron acceptance in tumor cells. In this regard,
the contents of U.S. Pat. No. 5,428,163 and the published methods
of C-Alkylation of phenols and their derivatives by Hudgens, T. L.
and Tumbull, K. D. are hereby incorporated by reference
From a physico-chemical and thermodynamic standpoint, the amount of
heat produced by uncoupling is proportional to the density and rate
of flux of electrons through the mitochondrial electron transport
chains. Such electron flux is initially reflected by the magnitude
of the electrochemical proton gradient across the inner
mitochondrial membrane. Those cells, tissues, organs and organisms
that are metabolically more active will generally have an increased
membrane potential and will respond with a greater amount of heat
production for a given dose and type of uncoupler. FIG. 12 lists
the six most "hottest" organs in the human body along with their
rates of blood flow and rates of heat production. The actual amount
of intracellular hyperthermia produced by an uncoupler is dependent
on the uncoupler dose, its relative potency and availability of
substrate such as glucose, glutamine, fatty acids or other
substances that produce NADH or FADH.sub.2. Oxygen and magnitude of
the mitochondrial proton electrochemical gradient
(.DELTA..mu.H.sup.+) are additional factors that determine the
amount of heat that can potentially be released by an uncoupler.
Among all the constituents, .DELTA..mu.H.sup.+ is the most
clinically important. .DELTA..mu.H.sup.+ is composed of the
transmitochondrial membrane potential [.DELTA..PSI. (charge
difference)] and pH gradient [.DELTA. pH (H.sup.+ concentration
difference)], .DELTA..mu.H.sup.+=F.DELTA..PSI.-2.3RT.DELTA.pH,
where, F=Faraday Constant, R=Gas Constant, and T=degrees Kelvin.
Thus, .DELTA..mu.H.sup.+ represents the potential amount of heat
that can be liberated by an uncoupler when 1 mole of H.sup.+ is
dissipated through the inner mitochondrial membrane. This potential
heat energy is normally expressed in units of millivolts (mV) and
is called the protonmotive force,
.DELTA.p=.DELTA..mu.H.sup.+/F=.DELTA..PSI.-2.3(RT/F).DELTA.pH. In
vivo, .DELTA.pH is generally 1 unit or less so that 75% or more of
the total .DELTA.p is comprised of .DELTA..PSI.. Consequently, the
intracellular heat produced by an uncoupler can be estimated by the
mitochondrial membrane potential (.DELTA..PSI.) alone.
Knowing the .DELTA..PSI. is of practical importance because biopsy
specimens may be incubated with cationic organic probes to estimate
the .DELTA..PSI. and the degree of differential heating that will
occur between normal and transformed tissues. Dyes such as
rhodamine 123, mitotracker green, calcein plus Co.sup.++,
3,3.sup.1-dihexyloxacarbocyanine, triphenylmethylphosphonium,
JC-1,5,5.sup.1,6,6.sup.1-tetrachloro-1,1.sup.1,3,3.sup.1-tetraethylbenzim-
idazolocarbocyanine, etc., all have an affinity for a negative
mitochondrial .DELTA..PSI.. Based on the amount of cationic dye
uptake, the membrane potential of specific tissue, tumors, and
cells may be determined through the Nernst equation:
.DELTA..PSI.=-(RT/F) ln(C.sub.in/C.sub.out). Which at physiologic
conditions and 37.degree. C. is =-61 log(C.sub.in/C.sub.out), where
C.sub.in/out is the concentration of the probe inside or outside
the mitochondria and plasma membrane. By example, a 10 to 1
gradient=-60 mV, 100 to 1=-120 mV. Uncouplers dissipate the
.DELTA..PSI., generate heat and release or prevent uptake of
cationic dyes. Six years of systematic measurement of mitochondrial
membrane potentials have been performed on human and mammalian
cells, including some 200 cell types derived from human malignant
tumors of kidney, ovary, pancreas, lung, adrenal cortex, skin,
breast, prostate, cervix, vulva, colon, liver, testis, esophagus,
trachea and tongue. Based on this exhaustive study, a .DELTA..PSI.
difference of at least 60 mV is known to exist between normal
epithelial cells and carcinoma cells. This is significant for the
present invention in that uncoupling or "short circuiting" a 60 mV
potential across a 5-nm mitochondrial membrane would be equivalent
to the amount of heat generated by short circuiting 120,000 V
across 1 centimeter. By exploiting or increasing the membrane
potential between normal and transformed cells the rate of
intracellular heat production by an uncoupler can be selectively
increased in target tissues.
In order for uncoupler induced intracellular hyperthermia to be of
therapeutic benefit, the development of thermotolerance is also
taken into account in practicing this invention. Mammalian cells
and prokaryotes acclimate and acquire transient resistance or
thermotolerance to gradual or non-lethal hyperthermia. Such
adaptation is believed to occur through increased synthesis of
highly conserved groups of proteins known as heat shock proteins
(HSP). The amount of HSP present in tissues, cells and organisms
subjected to non-lethal heat, or other forms of prolonged metabolic
stress, is proportional to their survival at higher temperatures.
In general, thermotolerance develops after 3 to 4 hours of
continuous hyperthermia, peaks in 1 to 2 days and decays back to
normal thermosensitivity within 3 to 4 days. Thermotolerance is
known to alter lethality of hyperthermia by as much as 2.degree. C.
increase or double the heating time required to achieve the same
temperature-cytotoxic effect. Such adaptive thermoresistance by
human tumors is problematic for continuous or fractionated
cytotoxic treatment with hyperthermia. Induction heating times with
the present invention are therefore kept to a minimum of 1 to 2
hours. Further, the uncoupler induced cytotoxic hyperthermia in the
present invention induces relative tissue hypoxia, lowers
intracellular pH and limits the production of ATP, all of which
repress the development of thermotolerance. Low doses of uncoupler,
which produce gradual heating can be used to induce HSP synthesis
and promote thermotolerance.
Determining the amount of DNP in mg/kg of body weight required to
produce the desired level of cytotoxic hyperthermia in a safe and
efficacious manner is established from the thermal equivalents
(Kcal) of oxygen consumed (V0.sub.2), and the known average
specific heat capacity of the human body. It is known that at
standard temperature and barometric pressure, 1 liter of oxygen
consumed per minute (VO.sub.2) generates approximately 4.862 Kcal.
It is also known that the average specific heat capacity of humans
is about 0.83 of that required to raise 1 gm of H.sub.20 1.degree.
K4.184 J, a heat capacity of 3.47 J g K.sup.-1. An initial estimate
of the total energy required to be generated by DNP to induce
41.0.degree. C. hyperthermia in 1 hour may be very simply
determined from the above and customized for a specific patient as
outlined below:
Patient Characteristics
Body weight 70 kg
Resting V0.sub.2 0.25 L/min
Basal energy expenditure 73.1 Kcal/hr (1754.4 Kcal/24 hrs.)
Basal core temperature 37.0.degree. C.
Target temperature 41.0.degree. C.
Required Energy to Raise Temperature to Target Level in 1 Hour
(Weight in grams=70.times.10.sup.3) (human specific heat=3.47 J g
K.sup.-1) (Temperature increase=41.0.degree.-37.0.degree.
C.).about.0.97.times.10.sup.6 J. Since 1 J=4.184.times.10.sup.-4
Kcal, a total power input of about 232 Kcal would be required to
raise the temperature of the patient to the objective level in 1
hour less that amount of heat generated by a heated metabolism
outlined below.
Increase in Metabolic Rate/Heat Production with Increase in Body
Temperature
The basal metabolic rate (BMR) is known to increase in patients
with endogenous fevers by approximately 7% for each 0.5.degree. C.
rise in temperature. This is graphically depicted in FIG. 11a. As a
result, the increase in BMR relative to the temperature will in
itself assist in achieving the objective level during the induction
phase by the following equation:
BMR.sub.Tcore=73.1.times.1.07.sup.(Tcore-37)/0.5
Thus, at 41.0.degree. C. the metabolic rate will be 134.4 Kcal/hr,
61.3 Kcal/hr above the basal energy expenditure level. This
increase in metabolic rate will therefore reduce the initial energy
required to heat the patient by approximately 61 Kcal over the 1
hour timeframe.
Initial Net Energy Input Required to Reach Target Temperature in 1
Hour
232 Kcal-61 Kcal (by increased BMR)=171 Kcal
Required Increase in Initial V0.sub.2 to Obtain 171 Kcal Heat
Input
Since the Kcal equivalent for 1 liter of oxygen consumed per minute
is 4.862, then the initial increase in VO.sub.2 required to
generate 171 Kcal can be calculated as follows: Heat in
Kcal/min=V0.sub.2.times.4.862. Since the individual patient has a
resting V0.sub.2 of 0.25 l/min which =73.1 Kcal/hour BMR, then
X(V0.sub.2)=171 Kcal, or X=0.25.times.171/73.1 An initial minimal
increase in V0.sub.2 to approximately 0.60 l/min is required. DNP
Dosage Required to Increase V0.sub.2 to 0.60 l/min
The individual DNP dosage (mg/kg) required to produce an increase
in oxygen consumption to 0.60 l/min so as to achieve a 171 K/cal
heat output is accomplished in the following fashion: (1) DNP is
prepared in a 200 mg/100 ml sterile aqueous solution. If not fully
dissolved, it can be brought into solution by buffering with 1%
NaHCO.sub.3, the pH must be kept below 8 to avoid hydrolysis; (2)
the dose of DNP for each intravenous infusion can vary from 0.5 to
4 mg/kg and will depend on the clinical situation, as well as the
initial and subsequent increases in the metabolic rate (V0.sub.2).
In an especially preferred embodiment, the patient is given an
initial dose of DNP no greater than 1 mg/kg intravenously, infused
over no less than a 2 minute period. Within approximately 10-15
minutes, a minimum of a 15% increase in V0.sub.2 will occur. The
V0.sub.2 will continue to increase until a plateau is reached
within an additional 5 to 10 minutes. After a 5 minute plateau in
V0.sub.2, a subsequent dose of either 0.5, 1, 2, 2.5, or 3.0 mg/kg
DNP is administered and V0.sub.2 is again increased until a desired
plateau is reached. Additional infusions of DNP or other
medications are administered under clinical parameters of V0.sub.2,
respiratory rate, pulse rate, blood pressure, urine output, cardiac
output, core temperature, and clinical status of the patient so as
to maintain safe and effective control of heating. If heat
dissipating mechanisms are neutralized, measurable increases in
core temperature will occur approximately 20 to 30 minutes after an
increase in the V0.sub.2. FIG. 13 illustrates the increases in
V0.sub.2 associated with repeated infusions of DNP.
Medications which increase the overall metabolic rate, or that of
specific target tissues, and have short half-lifes can be utilized
to increase the relative activity of DNP or other uncouplers to
further adjust V0.sub.2 and heat production. Examples of such
medications are almost limitless because any drug, hormone or
biologic response modifier that causes changes in enthalpy (heat
content) during the course of its intracellular chemical and
biophysical activity and interaction in the life cycle of
biological cells can be utilized. A few illustrative examples
include glucagon (half-life of 9 minutes in plasma), arbutamine
(half-life 10 minutes), dobutamine (half-life 2 minutes), and
vasopressin (half-life 5 minutes). Various amino acids and fatty
acids, e.g., glutamine, proline, octanoate, etc., increase V0.sub.2
by translocating reducing equivalents into the mitochondrial matrix
via the malate-aspartate shuttle, B-oxidation or proline
metabolism. Agents such as methylene blue (tetramethylthionine),
ubiquinone, menadione, hematoporphyrin, phenazine methosulfate,
2,6-dichlorophenolindophenol, coenzyme Q1, CoQ2, or their analogs
duroquinone and decylubiquinone, etc., can increase heat and/or
free radical production by acting as artificial electron acceptors.
Such agents, and numerous others, can be co-administered with DNP
or other uncouplers to effectively increase the enthalpy changes in
the entire organism or specific targeted tissues.
Minimizing Heat Loss and Temperature Control
Increased radiative and evaporative heat loss from man are the two
most dominant thermoregulatory mechanisms for cooling the body. The
body's methods of adjusting heat loss are vasoconstriction and
vasodilation in the skins blood vessels. Radiation can account for
60% of the heat loss generated by the body, while evaporation by
sweating at 1.0 liter/hour can represent a potential heat loss of
about 1,000 Kcal/hour. By far, sweating and evaporation is the
principal mechanism that dissipates heat under conditions that
induce large heat gains. Depending on the clinical circumstances,
heat loss due to evaporation, as well as radiation, can be managed
and controlled by a variety of methods including, but not limited
to, using vasoconstricting agents, placing the patient in a scuba
diving wet suit, humidified survival suit, or enveloping the
patient in a water soaked blanket covered or containing a
polyethylene lining to prevent evaporative heat losses. Use of room
ultrasonic nebulizers to induce continuous mist and high humidity
is also known to prevent evaporative heat losses. Evaporative and
radiant heat loss from the cranium is controlled by appropriate
head gear, shower caps and/or wet towels. Control of local air
velocities and management of surroundings as to temperature,
emissivity, drafts, and convection currents are important to avoid
large heat losses. In those clinical circumstances where total body
hyperthermia is required, failure to adequately control body heat
loss will necessitate using higher doses of DNP and induce a
greater metabolic stress upon the patient.
If the core target temperature is exceeded or continues to rise
after the target temperature is achieved, exposure of an extremity
or body surface for a brief interval will permit sufficient heat
loss to lower the core temperature to the target range. At target
temperatures of 39-41.degree. C., residual uncoupling by DNP will
continue for approximately 3 hours. Heat production as a byproduct
of glycolysis, and heated metabolism further maintains body heat
content and compensates for any heat loss. Therefore, target
plateau temperatures can be regulated with a large margin of safety
and with little to no additional use of uncoupler. Therapy is
terminated by removing the vapor barrier from the patient.
Evaporative and radiant heat loss from the patient generally
produces a fall in core temperature of about 2-2.5.degree. C. in
about 20-30 minutes. Obese patients and those with compromised
thermoregulatory systems experience a slower falloff in
temperatures.
Patient Monitoring, Fluid Support and Evaluation During
Treatment
Placement of physiologic monitoring sensors, intravenous fluids,
supplemental oxygen (4 l/min) and optional oral diazepam sedation
(5-10 mg) is initiated prior to treatment. Patients receive 0.85 to
1.0 liter of intravenous (IV) 5% dextrose in 0.25 normal saline per
hour alternated with 5% dextrose in 0.5 normal saline plus 7.5 to
10 meq of KCl per liter to insure a urinary output of no less than
1 ml/kg/hr. Oxygen consumption, caloric expenditure, rectal core
temperature, cardiac rhythm, blood pressure, heart rate and
respiratory rate are continuously displayed, monitored by a trained
member of the treatment staff. The data is automatically downloaded
into a computer every 20 seconds to 3 minutes for the entire
procedure and immediately re-displayed on computerized graphs and
charts. Two hours after treatment and 48 hours post-treatment,
serum chemistries and hematologic profiles are repeated. A typical
patient flow chart is depicted in FIG. 14.
Treatment of Excessive Heating and Antidotes
In those rare instances when too much uncoupler is administered or
the metabolic rate of the patient unexpectedly increases and
V0.sub.2, hyperthermia, pulse rate and patient fatigue ensue,
appropriate supportive measures of cooling, intravenous hydration
and administration of specific medication should be instituted.
Cooling should be instituted by uncovering the patient, spraying
with tepid water and fanning with an industrial grade fan. If
cooling is inadequate, surface, axillary and groin ice packs and
intravenous cold glucose solutions should immediately be
considered. Bicarbonate, 1-2 mEq/kg should be administered in the
absence of blood gas analysis. Urine output of >1 ml/kg/hour
should always be maintained to avoid pre-renal azotemia and
oliguria secondary to possible rhabdomyolysis and myoglobinuria.
Mannitol should be administered if urine output is inadequate.
Hypoglycemia should immediately be corrected with 50% saturated
intravenous glucose. If severe or persistent hypermetabolism
ensues, rectal propylthiouracil-1,000 mg, hydrocortisone (100 mg q
6 h) or dexamethasone 2 mg q 6 h intravenously and/or sodium iodide
as 1 g sodium ipodate (contrast agent) should be administered
intravenously to induce iatrogenic hypothyroidism. The decreased
metabolic rate will dramatically reduce the physiologic response to
DNP. Patient agitation and restlessness can be avoided by
appropriate IV or IM dose of diazepam. Salicylates are of no value
and may contribute to further uncoupling. Medications that reduce
sweating, e.g., tricyclic antidepressants, antihistamines,
anticholinergics, phenothiazines, or decrease vasodilation, e.g.,
sympathomimetics, .alpha.-agonists, or decrease cardiac output,
e.g., diuretics, beta-blockers or induce hypothalamic depression,
e.g., neuroleptics, .alpha.-blockers, opioids, etc., should be
avoided prior, during and immediately after treatment with
uncouplers.
The hypermetabolic and hyperthermic activity of DNP can further
specifically be reduced by using calcium channel blockers such as
nifedipine, verapamil and others, in intravenous doses that do not
cause a drop in blood pressure or induce cardiac arrhythmias.
Dihydrobenzperidol (a neuroleptic drug with
.alpha..sub.1-adrenergic properties) can also be used to cause
similar, significant reductions in DNP induced hypermetabolism and
hyperthermia. Dosages of these anti-DNP agents are titrated in 5 mg
to 30 mg increments and can be given either by mouth or
intravenously. In those cases where DNP appears to decrease
electrical conduction or cause EKG conduction abnormalities,
Coenzyme Q10, in doses of 50 mg/kg, can be used to restore normal
electrical activity.
Patient Selection and Pretreatment Evaluation
It is imperative that in the practice of this invention, patients
be selected and evaluated prior to treatment. Recommended patient
inclusion and exclusion criteria includes: (1) patients have a
definitive histopathologic or other laboratory confirmed diagnosis
of their disease; (2) the disease or condition should be responsive
to intracellular hyperthermia treatment; (3) patients should have a
Karnofsky score of 70% or greater; (4) not be pregnant; (5) weight
should be within 45% (+/-) of ideal body weight and patients must
weigh at least 35 kg; (6) there should be no history or findings of
anhidrosis, scleroderma, ectodermal dysplasia, Riley-Day Syndrome,
arthrogryposis multiplex, extensive psoriasis, serious
dysrhythmias, malignant hyperthermia or neuroleptic malignant
syndrome, pheochromocytoma, hypocalcemia, repeated episodes of
hypoglycemia, chronic or recurrent venous thrombosis, alcoholism,
renal failure, cirrhosis, untreated hyperthyroidism, anaphylaxis
associated with heat or exercise-induced cholinergic type
urticaria, exercise or heat induced angioedema, schizophrenia,
catatonia, seizure disorders, emotional instability, Parkinson's
disease, brain irradiation, cystic fibrosis, unstable angina
pectoris, congestive heart failure, patients with cardiac
pacemakers, severe cerebrovascular disease, spinal cord injury,
severe pulmonary impairment, hereditary muscle disease such as
Duchenne type muscular disease, central core disease of muscle,
myotonia congenita, King-Denborough syndrome, Scwanry-Jampol
syndrome, or osteogenesis imperfecta; (6) no immediate use of drugs
that impair the body's heat dissipation mechanisms such as
phenothiazines, anticholinergics, antihistamines,
antiparkinsonians, glutethimide, hallucinogens, lithium, cocaine or
other illicit drug use, monamine oxidase inhibitors,
sympathomimetics, phencyclidine, opioids, phenylephrine, INH,
tricyclic antidepressants, withdrawal from dopamine agonists, or
cardiovascular drugs that clinically impair cardiac output or
thermoregulatory vasodilation such as high doses of
.beta.-blockers, vasodilators, or calcium channel blockers; and,
(7) the patient should not be anemic or otherwise have a reduced
oxygen absorbing, carrying or utilizing capacity.
Pretreatment evaluation should include a complete medical history
and physical examination focused on the selection criteria listed
above. Laboratory evaluation should include pulmonary function
tests-if indicated, full hematological survey with hemostatic
profile, EKG, liver function tests, serum biochemical profile,
thyroid panel, serum creatinine, calcium, phosphate, and stress-EKG
or exercise-multigated radionucleotide ejection scan on patients
whose cardiac ejection fraction is suspect not to be greater than
45% with probable deterioration on exercise. While clinical
exceptions to entry laboratory values may exist, the following
laboratory data should be a benchmark guide for initiation of
treatment: hemoglobin>=11.0 g/dl for men and >=10.0 g/dl for
women, platelet count>=75.00 platelets/mm.sup.3,
bilirubin<=2.times.ULN (ULN=upper limit of normal), ALT
(SGPT)<=2.times.ULN, AST (SGOT)<=2.times.ULN, pancreatic
amylase<1.5.times.ULN, neutrophil count>=1,000
cells/mm.sup.3. Serum electrolytes and K.sup.+ should be well
within normal limits, as hypokalemia decreases muscle blood flow,
cardiovascular performance, and sweat gland function.
More generally, the method outlined above is to be tailored to an
individual patient. As set forth above, the DNP may be administered
by intravenous infusion. Alternatively, the route of administration
may also be orally, rectally or topically. The frequency and
optimal time interval between administrations is individualized and
determined by measuring V0.sub.2, as well as other parameters. For
example, various laboratory, x-ray, CAT scan, MRI, PET scan, HIV
load, CD4+ lymphocyte counts, HSP expression, prostatic specific
antigen (PSA) and other surrogate markers of clinical outcome can
establish the VO.sub.2, frequency and duration of therapy. One
treatment, or treatments as frequent as every day, or every other
day, as far apart as 1 year or longer may be required for sustained
beneficial results.
The optimal VO.sub.2, temperature, duration, and frequency between
treatments will probably vary from patient to patient and the
specific disease or condition being treated. One skilled in the art
would be able to modify a protocol within the present invention, in
accordance with standard clinical practice, to obtain optimal
results. For example, the HIV relationships between viral load,
CD4.sup.+ lymphocyte counts, presence of opportunistic infections
and clinical status of the patient can be used to develop more
optimal regimes of DNP administration. Applicants' studies have
revealed that the methods of the present invention can be effective
in the diagnosis and treatment of a wide range of disease states
and conditions in which uncoupler induced hypermetabolism,
hyperthermia, oxidative stress and their sequela, play a beneficial
role. To those skilled in the art, it is also encompassed that a
variety of different veterinary, as well as medical, applications
for treatment and diagnosis can be practiced with the present
invention.
It is envisioned that DNP, or other uncouplers, may also be
administered with other compounds used to treat infectious,
malignant or other diseases. Examples of other agents include
antifungal, antibacterial, antiviral or anti-neoplastic drugs, cell
differentiating agents, and, various biologic response modifiers.
Examples of anti-fungal agents include Amphotericin B,
Griseofulvin, Fluconazole (Diflucan), Intraconazole, 5
fluoro-cytosine (Flutocytosine, 5-FC), Ketatoconazole and
Miconazole. Examples of anti-bacterial agents include antibiotics,
such as those represented from the following classifications: beta
lactam rings (penicillins), macrocyclic lactone rings (macrolides),
polycyclic derivatives of naphthacenecarboxamide (tetracyclines),
amino sugars in glycosidic linkages (aminoglycosides), peptides
(bacitracin, gramicedin, polymixins, etc.), nitrobenzene
derivatives of dichloroacedic acid, large ring compounds with
conjugated double bond systems (polyenes), various sulfa drugs
including those derived from sulfanilamide (sulfonamides,
5-nitro-2-furanyl compounds (nitrofurans), quinolone carboxylic
acids (nalidixic acid), fluorinated quinilones (ciprofloxan,
enoxacin, ofloxacin, etc.), nitroimidazoles (metroindazole) and
numerous others. These antibiotic groups are examples of preferred
antibiotics, and examples within such groups include: peptide
antibiotics, such as bacitracin, bleomycin, cactinomycin,
capreomycin, colistin, dactinomycin, gramicidin A, enduracitin,
amphomycin, gramicidin J, mikamycins, polymyxins, stendomycin,
actinomycin; aminoglycosides represented by streptomycin, neomycin,
paromycin, gentamycin ribostamycin, tobramycin, amikacin;
lividomycin beta lactams represented by benzylpenicillin,
methicillin, oxacillin, hetacillin, piperacillin, amoxicillin and
carbenacillin; lincosaminides represented by clindamycin,
lincomycin, celesticetin, desalicetin; chloramphenicol; macrolides
represented by erythromycins, lankamycin, leucomycin, picromycin;
nucleosides such as 5-azacytidine, puromycin, septacidin and
amicetin; phenazines represented by myxin, lomofungin, iodin;
oligosaccharides represented by curamycin and everninomycin;
sulfonamides represented by sulfathiazole, sulfadiazine,
sulfanilimide, sulfapyrazine; polyenes represented by
amphotericins, candicidin and nystatin; polyethers; tetracyclines
represented by doxycyclines, minocyclines, methacylcines,
chlortetracyclines, oxytetracylcines, demeclocylcines; nitrofurans
represented by nitrofurazone, furazolidone, nitrofurantoin, furium,
nitrovin and nifuroxime; quinolone carboxylic acids represented by
nalidixic acid, piromidic acid, pipemidic acid and oxolinic acid.
The Encyclopedia of Chemical Technology, 3rd Edition, Kirk-Othmer,
editors, Volume 2 (1978), which is hereby incorporated by reference
in its entirety.
Antiviral agents that can be used with DNP include: interferons
.alpha., .beta. and .gamma., amantadine, rimantadine, arildone,
ribaviran, acyclovir, abacavir, vidarabine (ARA-A)
9-1,3-dihydroxy-2-propoxy methylguanine (DHPG), ganciclovir,
enviroxime, foscarnet, ampligen, podophyllotoxin, 2,3-dideoxytidine
(ddC), iododeoxyuridine (IDU), trifluorothymidine (TFT),
dideoxyinosine (ddi), d4T, 3TC, zidovudine, efavirenz, protease
inhibitors such as indinavir, saquinavir, ritonavir, nelfinavir,
amprenavir, etc., and specific antiviral antibodies.
Anti-cancer drugs that can be used with DNP include, but are not
limited to, various cell cycle-specific agents represented by
structural analogs or antimetabolites of methotrexate,
mercaptopurine, fluorouracil, cytarabine, thioguanine, azacitidine;
bleomycin peptide antibiotics, such as podophyllin alkaloids
including etoposide (VP-16) and teniposide (VM-26); and various
plant alkaloids such as vincristine, vinblastine, and paclitaxel.
Anti-neoplastic cell cycle-nonspecific agents such as various
alkylating compounds such as busulfan, cyclophosphamide,
mechlorethamine, melphalan, altretamine, ifosfamide, cisplatin,
dacarbazine, procarbazine, lomustine, carmustine, lomustine,
semustine, chlorambucil, thiotepa and carboplatin. Anticancer
antibiotics and various natural products and miscellaneous agents
that can be used with DNP include: dactinomycin, daunorubicin,
doxorubicin, plicamycin, mitomycin, idarubicin, amsacrine,
asparaginase, quinacrine, retinoic acid derivatives (etretinate),
phenylacetate, suramin, taxotere, tenizolamide, gencytabine,
amonafide, streptozocin, mitoxanthrone, mitotane, fludarabine,
cytarabine, cladribine, paclitaxel (taxol), tamoxifen, and
hydroxyurea, etc.
DNP can also be administered with various hormones, hormone
agonists and biologic response modifying agents which include, but
are not limited to: flutamide, prednisone, ethinyl estradiol,
diethylstilbestrol, hydroxyprogesterone caproate,
medroxyprogesterone, megestrolacetate, testosterone,
fluoxymesterone and thyroid hormones such as di-, tri- and
tetraiodothyroidine. The aromatase inhibitor, amino glutethimide,
the peptide hormone inhibitor octreotide and gonadotropin-releasing
hormone agonists such as goserilin acetate and leuprolide can also
be used with DNP. Biologic response modifiers such as various
cytokines, interferon alpha-2a, interferon alpha-2b,
interferon-gamma, interferon-beta, interleukin-1, interleukin-2,
interleukin-4, interleukin-10, monoclonal antibodies
(anti-HER-2/neu humanized antibody), tumor necrosis factor,
granulocyte-macrophage colony-stimulating factor,
macrophage-colony-stimulating factor, various prostaglandins,
phenylacetates, retinoic acids, leukotrines, thromboxanes and other
fatty acid derivatives can also be used with DNP.
The use of this invention should be under the strict direction of a
qualified and specialized treatment team to insure safety and
effectiveness. The treatment team remains with the patient
throughout the procedure to insure that safe and controlled dosages
of an uncoupler are administered by monitoring real time changes in
V0.sub.2, metabolic rate, temperature, respiratory rate, heart
rate, urine output and clinical status of the patient. This
invention is practiced in controlled steps so as to attain a
predetermined V0.sub.2 and plateau of heating time for a particular
disease or condition. For example, in cases were heat dissipation
mechanisms do not have to be blocked, the specialized team will
periodically recheck V0.sub.2, heart rate, blood pressure, CAT
scan, MRI, etc., and other laboratory and clinical parameters to
insure continued safety and efficacy of DNP therapy. It is
preferred that the specialized team undergo a training period in
the use of this invention prior its administration to human
patients.
The present invention is further illustrated by reference to the
following examples, which illustrate specific elements of the
invention but should not be construed as limiting the scope of the
invention.
EXAMPLE 1
Method of Using DNP with Glucagon to Treat Parasitic Infections,
Hydatid Disease of the Liver
History: A 52 year old white Swiss male, European fox hunting dog
trainer, presented with right upper quadrant pain and vomiting.
Past history revealed he had hepatic "cyst" surgery 2 years ago.
Preoperatively, he was treated with albendazole. Only one dose of
albendozale was given because of a "near death" anaphylactic
reaction. He denied history of weight loss, pulmonary, cardiac,
neurologic or thermoregulatory problems. There was no history of
alcohol abuse or medication use. The patient was adamantly opposed
to any further surgery or treatment with albendazole or
mebendazole.
Physical Examination Weight=90 Kg; height=177.8 cm; BP=140/80;
HR=76 & reg; Resp.=18 min; T=37.0
An old well healed scar consistent with prior hepatic surgery was
present. Physical exam otherwise was unremarkable.
Laboratory studies: EKG, chest X-ray, blood panel, including serum
electrolytes, thyroid studies and liver function tests were within
normal limits (WNL). A complete blood count was unremarkable except
for 20% eosinophilia. Ultrasound and nuclear magnetic resonance
revealed 4, 2 to 3 cm. in diameter, cysts in the right middle lobe
of the liver and a solitary 2 cm semi-solid medullary cyst in the
neck of the right humerus. ELISA serology showed a diagnostic titer
for hydatid disease. Review of previous surgical liver pathology
reports revealed a cestode compatible with Echinococcus
multilocularis.
Clinical assessment and treatment evaluation: The patient had no
historical or physical contraindications to DNP induced
hyperthermia. Conventional therapy of hydatid disease is either
surgical resection or medical therapy with albendazole for 4 weeks.
Hydatid bone cysts are not amenable to surgery and respond poorly
to standard medical therapy. Echinococcus multilocularis
protoscoleces and the germinal membranes of hydatid cysts are known
to be irreversibly destroyed by heating at 41.degree. C. for 15
minutes. Human liver and hepatocytes can withstand artificial
temperatures of 42.degree. C. for as long as 20 hours without
irreversible damage. Acute glucagon treatment is known to
preferentially stimulate hepatocyte mitochondrial V0.sub.2. Rates
of hepatocyte uncoupled V0.sub.2 are also know to be stimulated up
to 100% in less than 6 minutes after the hormonal action of
glucagon. Acute glucagon treatment has been shown to selectively
increase the pH gradient across hepatocyte mitochondrial membranes.
Thus, it can be empirically presumed that any increase in V0.sub.2
from glucagon administration causes increased thermogenesis,
predominantly in the liver.
Pretreatment protocol: the patient was given 10 mg diazepam by
mouth and dressed into a modified wet suit. The wet suit was cut
lengthwise at the arms and legs. Velcro strappings were attached at
the cuttings for closure, rapid removal or exposure of the limb(s).
After placement of monitoring sensors, he was started on IV fluids
of 5% dextrose, 0.5 normal saline with 7 meq K.sup.+, infused at an
initial rate of 12 cc/kg/hr. Evaporative heat loss from the head
was minimized by a plastic shower cap and towels. A 401AC
temperature probe (YSI Incorporated, Yellow Springs, Ohio) was
inserted 11 cm. into the rectum. The probe was connected to a Model
4600 telethermometer (YSI 4600 Precision Thermometer) and readings
within 0.1.degree. C. were continuously displayed and recorded at
baseline and during treatment on Hewlett-Packard (HP) computer
systems with customized software developed by MR&S (Manalapan,
N.J.). A TEEM 100 Metabolic Analysis System (AeroSport Inc., Ann
Arbor, Mich.), with a modified face mask and oxygen delivery system
(38-40% 0.sub.2 saturation) for patient comfort and increased
accuracy, was attached to the patient. Oxygen consumption
(V0.sub.2), carbon dioxide production (VC0.sub.2), expired air
volume (V.sub.E), heart rate (HR), and Kcal of heat produced were
measured in 20 second intervals and extrapolated to minute or
hourly rates. All patient data was monitored in real time,
continuously displayed at baseline and during treatment and
recorded on HP computer systems with customized software from
MR&S (Manalapan, N.J.).
Treatment procedure: After baseline recordings of 10 minutes, the
required amount of DNP to raise the initial V0.sub.2 to achieve a
temperature in the patient of 40.degree. C. was calculated as
described under "DNP dosage required to increase V0.sub.2". The
patient was given an initial dose of 1 mg/kg of DNP, infused
intravenously over a 3 minute period. After the V0.sub.2 stabilized
at 40% above baseline, an additional DNP infusion of 3 mg/kg was
given. Upon attaining a stable V0.sub.2, 0.5 mg of glucagon was
administered intravenously. After this stabilization of V0.sub.2, a
glucagon drip was variably infused from 0.5 to 5 mg/kg/hour to
additionally control V0.sub.2 and selectively augment heat
production in the liver. The treatment procedure was discontinued
after the patient was maintained at a rectal body temperature of
40.degree. C. for about 1 hour; The wet suit was opened and head
covering removed. After the patient's body temperature reached
38.degree. C., the Foley catheter was removed and intravenous
fluids were discontinued. Evaporative and radiant heat loss lowered
the body temperature to a normothermic level within 30 minutes. No
immediate or delayed post-treatment toxicity was encountered.
Monitored patient parameters are shown in FIG. 15.
Treatment outcome: Serial imaging studies revealed hepatic and bone
cyst shrinkage with increased density at 2 and 4 weeks post
treatment. Repeat magnetic resonance imaging at 4 months showed
complete cyst disappearance in the liver and bone.
EXAMPLE 2
Method of Using DNP to Treat Viral Infections, HIV Disease
History: A 38 year old white male, past intravenous heroin addict,
was diagnosed approximately 8 years ago with HIV by ELISA and
positive Western blot for HIV p24 and gp41 antigens after
presenting with weight loss and thrush. His history included
repeated treatment for candidiasis, pneumocystis carinii, and
various subcutaneous abscesses. Past medications included
sulfamethoxazole, ketoconazole, fluconazole, zidovudine, didanosine
and various other antibiotics. For the past year and a half he has
been on highly active antiretroviral therapy (HAART) with various
HIV protease inhibitors combined with thymidine, purine or cytosine
nucleoside and nonnucleoside inhibitors. He was unable to tolerate
nelfinavir because of diarrhea. Ritonavir caused intractable
vomiting and abdominal pain. Current medications include indinavir,
zidovudine and lamivudine. Review of the most recent viral load
(VL) and CD4+ lymphocyte counts showed an initial drop in plasma
HIV RNA (copies/ml) from 200,000 to 2,000 over a 12 week period
with the VL rebounding back to 200,000 at week 16. CD4+ lymphocyte
counts have remained between 100 to 200 cells/mm.sup.3.
Approximately 5 months ago he was treated for oral and
endobronchial Kaposi's sarcoma (KS) with liposomal daunorubicin
followed by liposomal doxorubicin. He denied treatment with
vincristine or bleomycin. There is no history of recent diarrhea,
recent weight loss, hemoptysis, shortness of breath on moderate
exertion, or cardiac problems. There has been no illicit drug use
over the past 2 years. The patient stated no combination of HAART
has been able to lower his viral load and multiple side effects
from the drugs are limiting his compliance to take the medications.
There was no history of thermoregulatory problems.
Physical examination: weight=60 Kg; height=155 cm; BP=128/72;
Resp=20; T=38.2.degree. C.; and, the pulse was 92 & reg. Exam
revealed asthenia and generalized enlargement of lymph nodes, some
2 to 3 cm in diameter in the axillary and inguinal regions. There
was diffuse oropharyngeal thrush. Beneath the thrush, the oral
cavity also contained several dark red plaque to nodular like
lesions on the hard palate and gingiva. The lesions did not blanch
on compression with the tongue blade. A crusted strawberry like
mass, 1 by 2 cm, was present at the anus. There were no neurologic
deficits or ocular lesions.
Laboratory studies: EKG, serum electrolytes, renal and liver
function tests were normal. Hematocrit was 35.5%, WBC was 9,900
with 81% neutrophils, 4 bands, 11 lymphocytes and 4 monocytes.
Platelets were 314,000/mm.sup.3. Viral load was 400,000 copies/ml
(Amplicor HIV Monitor test, Roche). A CD4.sup.+ T cell count was
quantified by flow cytometry at 250/mm.sup.3. He was antibody
positive for hepatitis C. Chest radiograph showed some bilateral
apical patchy opacities. Pulmonary function tests showed all
parameters, including forced expired volume, greater than 80% of
predicted. Karnofsky score was greater than 70. Normal and tumor
tissue biopsies, 3 to 6 mm in diameter, from the oral cavity and
anus were obtained. The tissues were equally divided, weighed and
placed in 4.degree. C. Ringers lactate solution. Histologically
confirmed normal and KS tissues were then subjected to
microcalorimetric measurements in a thermal activity monitor
(ThermoMetric, Jarfalla, Sweden). Recorded heat output (.mu.W/min)
was 8.2-8.5 times greater for the KS sarcoma lesions than
nontumorous oral mucosa tissues. Repeat measurements with biopsies
specimens in 30 uM DNP increased heat production in tumorous
tissues 20.5 times more than nontumorous specimens.
Clinical assessment and treatment evaluation: HIV and HIV-infected
T cells are known to be more sensitive to killing by heat than
uninfected lymphocytes. Susceptibility to heat killing is enhanced
with increased oxygen free radical production. Acute and
chronically infected cells have decreased levels of manganous
superoxide dismutase (MnSOD) activity. MnSOD is located exclusively
in mitochondria. Mathematical modeling of human HIV production and
CD4+ T cell turnover predicts that reducing both free virus and
actively infected cells by a minimum of 40% with 1 hour of
42.degree. C. therapeutic hyperthermia every third day will promote
recovery of the uninfected T-cell population. Human HIV studies
with extracorporeal hyperthermia of 41-42.degree. C. have reported
isolated cases of extended patient survival, elimination of
detectable virus, and improvement of Kaposi's sarcoma lesions. DNP
is known to generate intracellular hyperthermia and oxygen free
radicals from the level of the inner mitochondrial membrane.
Studies on in vitro inactivation of chronically HIV infected HUT-78
cells by various concentrations of DNP are graphically represented
in FIG. 16.
The patient has been and remains resistant to treatment with HAART.
Opportunistic infections with candida and Kaposi's sarcoma herpes
virus (KSHV, human herpesvirus type 8) causing his thrush and
Kaposi's sarcoma are comorbid conditions indicative of a worsening
prognosis. In spite of having AIDS with candidiasis and Kaposi's
sarcoma, the patient maintains good cardiac and pulmonary function.
There was no history of thermoregulatory problems. It was discussed
and agreed that hyperthermia treatments with core body temperatures
of 41.degree. C. would be administered on a daily or every other
day basis, as tolerated, for a minimum of 3 hours, not to exceed 5
hours.
Pretreatment protocol: all medications were stopped 2 weeks prior
to treatment. The patient refused taking diazepam, placement of a
Foley catheter and oxygen face mask. He dressed himself into a dry
cold water immersion suit (Stearns, ISS-590I, Universal Adult)
designed to prevent heat loss and modified for easy placement of
physiologic monitors. Equipment for measurement of heart rate,
temperature, carbon dioxide production and Kcal of heat produced
were conducted as outlined in Example 1. An oral breathing tube was
used to measure V0.sub.2 from room air. Urine output was measured
when the patient voluntarily urinated through a "Texas" catheter
(superficial condom tightly fitted around the head of the penis
with tubing connected to urine collection bag). The patient was
informed that hyperthermia would be administered as tolerated by
his stamina and monitored clinical parameters, not to exceed 5
hours, on a daily or every other day basis, for a total of 5
sessions.
Treatment procedure: Baseline reading for 5 minutes established an
average V0.sub.2 of 300 cc/min. An initial dose of 2 mg/kg of DNP
was administered over a 2 minute period. V0.sub.2 increased and
stabilized at 15 minutes at 340-380 cc/min. An additional 2 mg/kg
DNP infusion was given, the V0.sub.2 increased and stabilized at
610-630 cc/min. Body core temperature increased to 39.4.degree. C.
within 60 minutes. A gradual fall in blood pressure was noted at 90
minutes to 100/60 mm/Hg. Norepinephrine bitartrate (Levophed) was
given IV drip at a dose of 1 microgram/min. and adjusted to
maintain blood pressure at 130/80. Approximately 1 minute after
initiating the vasopressor, heart rate increased from 90 to 100 and
V0.sub.2 to 0.85 liters/min. Core body temperature increased within
20 minutes to 41.5.degree. C. V0.sub.2 was maintained at 1.0
liters/min. by lowering or increasing the dose of norepinephrine.
An additional infusion of 1 mg/kg DNP was given at hour 4 to
correct a dropping V0.sub.2. On occasions when the core temperature
increased above 41.6.degree. C., a lower extremity was exposed for
evaporative heat loss. The patient withstood the procedure without
any untoward effects for a period of 7 hours. The protocol was
repeated consecutively for 5 days without the additional use of
vasopressors.
Treatment outcome: Immediately after the first treatment oral
candidiasis improved by 50%. The oral and anal Kaposi's lesions
exhibited marked erythema with circumferential areas of blanching.
On the second day of treatment the KS erythema diminished. There
was no evidence of oral candidiasis on the 3.sup.rd day of therapy.
The anal tumor was crusted and approximately 60% diminished in size
on the 5.sup.th and last day of therapy. Lymphadenopathy
progressively decreased and was resolved at 2 weeks post-treatment.
At 30 days post-treatment, there was complete regression of both
oral and anal KS lesions. Repeat blood work on days of treatment
showed no significant hematologic, electrolyte, liver or kidney
changes from baseline. Viral load immediately after treatment day 5
showed 50,000 HIV-RNA copies/ml. HIV RNA was non-detectable at 4, 6
and 12 weeks post-treatment. CD4+ T cell lymphocyte counts
increased to 380-420 cells/mm.sup.3 by week 4 and remained stable
at week 6 and 12. FIG. 17 shows monitored patient parameters on
treatment day 1. FIG. 17a) shows changes in surrogate markers
immediately after treatment, weeks 4, 6 and 12.
EXAMPLE 3
Use of DNP to Treat Bacterial Infections, Lyme Disease
History: A 33 year old white female with a textbook case of Lyme
borreliosis related being bitten by a tick and developing a
pathognomonic erythema migrans on her right anterior thigh. The
rash resolved within two weeks but 3 months later she developed
verbal memory impairment, migratory arthritis of the knees, ankles
and tibias. Fibromyalgias, tachycardias and a left sided Bell's
palsy ensued. Constitutional symptoms of fatigue, malaise and
severe depression caused her to undergo psychiatric care for 11/2
years before she was definitively diagnosed with chronic Borrelia
burgdorferi infection. She was treated with ceftriaxone, 2 g
intravenously every 12 hours for 14 days. Four months after
apparent improvement she developed photophobia, headaches,
pronounced memory loss, depression, dysesthesias and a painful,
swollen left knee joint. Repeat ELISA, Western blot and DNA-PCR
were all positive for B. burgdorferi. Spinal tap showed pleocytosis
with positive antibody and PCR tests for neuroborreliosis. Over the
next year the patient received prolonged ceftriaxone, 2 g per day
intravenously for 3 months, and 3 individual short courses of oral
ciprofloxacin, minocycline, and azithromycin. Symptoms failed to
resolve. Two months after her last regimen of antibiotics a new
annular erythematosus eruption, suggestive of erythema migrans,
reoccurred on the right thigh and developed under her left axilla.
Doxycycline was instituted and the rash subsided. The patient
refused further antibiotic therapy because of associated
intractable diarrhea and has made tentative plans to undergo
"malariotherapy" in China.
Physical examination: weight=60 Kg; height=160 cm; BP=130/70; HR=86
& reg; resp=18; T=37.3.degree. C. Physical exam revealed a
swollen and tender left knee. A thin, atrophic hypopigmented area
of skin over the right thigh, typical of acrodermatitis chronica
atrophicans was present. Neurologic exam showed some verbal memory
deficit. There were bilateral, lower distal extremity
paresthesias.
Laboratory studies: EKG demonstrated a first-degree
atrioventricular block (PR internal>0.2 sec), some widening of
the QRS complex and Wenckebach periodicity. There were no dropped
beats. Left knee arthroscopy showed synovial hypertrophy with early
erosive arthritis. Synovial fluid analysis revealed a WBC of 50,000
cells/ml with 70% neutrophils and a positive DNA-PCR for Borrelia
burgdorferi. Biopsy sections of synovial tissue showed chronic
nonspecific synovitis. Warthin-Starry and silver staining histology
revealed spirochetal organisms consistent with Borrelia
burgdorferi. Lumbar puncture spinal fluid analysis showed
pleocytosis, elevated gamma globulin and positive PCR for B.
burgdorferi. Spinal fluid cultured for 2 months in
Barbour-Stoenner-Kelly medium was reported positive for B.
burgdorferi. Serum electrolytes, kidney, liver function and
hematologic studies were all within normal limits. The patient
underwent a stress EKG, attaining a maximum heart rate of 165 with
no evidence of arrhythmia or S-T segment depression.
Clinical assessment and treatment evaluation: Lyme disease is a
zoonosis caused by a slow growing pathogenic spirochete, Borrelia
burgdorferi. In various mammalian species, including man, these
organisms are known to invade heart, kidneys bladder, spleen and
brain. Borrelia spirochetes are very resistant to treatment with
antibiotics, especially if there is evidence of central nervous
system or joint involvement. Viable B. burgdorferi have been
isolated from antibiotic treated monolayers of fibroblasts.
Borrelia spirochetes are known to be facultative intracellular
pathogens in fibroblasts by laser scanning confocal microscopy.
Central nervous system tissue, joints, front chamber of the eye and
intracellular location can provide the Lyme spirochete with a
protective environment against antibiotic therapy and Borrelia
burgdorferi have been reliably cultured from patients with chronic
disease, even from those previously aggressively treated. This
patient has confirmed chronic CNS and joint Lyme disease in spite
of extensive antibiotic therapy.
The Lyme spirochete is irreversibly inactivated by heating at
40.degree. C. for 3 hours, 41.degree. C. for 2 hours or
41.5.degree. C. for 1 hour. Susceptibility of all strains of
Borrelia burgdorferi to penicillin and ceftriaxone is increased up
to 16-fold by elevation of temperature from 36.degree. C. to
38.degree. C. At 40.degree. C. Borrelia burgdorferi increases
expression of at least 12 heat shock proteins (HSP), most of which
are strongly immunogenic. The patient had no history of
thermoregulatory problems. She was informed that her body
temperature would be raised between 4.degree. to 41.degree. C. for
a period of 3 hours, the actual level and time under hyperthermia
would depend on her monitored clinical parameters.
Pretreatment protocol: the evening prior treatment the patient was
instructed not to eat and dress in cotton undergarments.
Approximately 4 hours prior to treatment 2 mg alprazolam was
administered by mouth. The patient dressed herself into a dry cold
water immersion suit (Stearns, previously described) with headgear.
Monitoring sensors, including EKG display, IV fluids and Foley
catheter were attached and the suit was zipped closed. The patient
opted for oxygen supplementation. The modified face mask was
connected to the TEEM 100 metabolic Analysis System for V0.sub.2
measurements. Data was recorded as previously described.
Treatment procedure: baseline recordings of 10 minutes showed a
V0.sub.2 of 220 cc/min., 3.7 cc 0.sub.2/kg/min. The patient was
infused with 1 mg/kg DNP over a 2 minute period. V0.sub.2 increased
and stabilized at 250 cc/min, 5.3 cc/kg/min. A second dose of 2.0
mg/kg was infused over a 2 minute period and the V0.sub.2 peaked at
400 cc/min, 8.8 cc 0.sub.2/kg/min. An additional dose of 1.0 mg/kg
DNP was given 30 minutes after the second dose. The V0.sub.2
increased and reached a stable plateau at 600 cc/min, 10.8
cc/kg/min. Rectal temperature continued to climb until a range of
40.2 to 40.6.degree. C. was reached at 70 minutes after the initial
dose. A fall in V0.sub.2 was noted at 90 minutes, a dopamine drip
at 2-3 mcg/kg/min was initiated. V0.sub.2 increased back to 680-710
cc/min. The temperature remained stable between 40.1.degree. C. and
40.6.degree. C. throughout the 3 hour plateau treatment period. The
patient periodically requested the V0.sub.2 monitoring mask be
removed during the hyperthermia treatment period. She was
accommodated with removal of the mask on two occasions for periods
not exceeding 10 minutes. The patient experienced no problems
during the procedure but was noticeably fatigued by hour 3. The
treatment was terminated 4 hours and 10 minutes after the initial
dose of DNP. Twenty five minutes after the patient was removed from
the neoprene survival suit, the rectal core temperature dropped to
38.5.degree. C. Normothermia was achieved approximately 60 minutes
after cessation of therapy and removal from the survival suit.
Approximately 6.5 to 7 hours after treatment the patient
experienced chills, an increase in oral temperature to 38.7 degrees
centigrade and malaise. IV fluids and the dopamine drip at 2
mcg/kg/min were restarted and the patient was closely observed. Her
symptoms subsided over 3 hours and by the next day she felt active
and hungry. It was surmised she may have experienced a delayed
Jarisch-Herxheimer reaction. The patients monitored treatment flow
chart is FIG. 18.
Treatment outcome: at two months follow-up the patient stated her
arthralgias, myalgias, malaise, fatigue and memory deficits have
disappeared. Lower extremity dysesthesias were no longer present.
EKG showed resolution of her first degree A-V block. The patient
was informed of her past positive cerebrospinal fluid positive
culture for the Lyme disease spirochete. It was suggested a repeat
spinal tap be performed for B. burgdorferi by PCR and culture. If
positive, the patient agreed she would be re-treated with both DNP
induced hyperthermia and intravenous ceftriaxone for maximum
synergism. Repeat spinal fluid analysis was normal, i.e., no
elevated protein, no detectable Borrelia DNA by PCR and no
pleocytosis. Three months later, spinal fluid culture on
Barbour-Stoenner-Kelly II medium was reported negative.
EXAMPLE 4
Method of Using DNP with Vasopressors and Chemotherapy to Treat
Neoplasia, Peritoneal Carcinomatosis
History: A 55 year old female presented with a distended abdomen
due to ascites. Laparotomy revealed peritoneal dissemination of a
malignancy with histological findings of an undifferentiated
adenocarcinoma, origin unknown.
Physical examination: weight=55 kg; height=154 cm; BP=140/90; HR=88
& reg; Resp=22; T=37.6.degree. C. The patient was a well
developed and well nourished Muslim female with a healing midline
laparotomy scar. Ballotable ascites was detected in the abdomen.
There was no lymphadenopathy.
Laboratory studies: laboratory examination of the ascitic fluid
showed high levels of amylase. She had a hemoglobin of 9.2. High
levels of amylase and tumor markers, including CA15-3, CA 125 and
CA72-4 were present in the serum. Blood chemistry, liver and kidney
function tests were within normal limits. Chest X-ray and EKG was
normal. MRI and ultrasound of the abdomen showed normal pancreas,
liver and atrophic ovaries, there were widespread nodular lesions
consistent with peritoneal carcinomatosis.
Clinical assessment and treatment evaluation: the patient had an
inoperable malignancy of unknown origin. Chemotherapy in such cases
is only of marginal survival benefit. Hyperthermia, combined with
chemotherapy has been shown to be synergistic with increased tumor
response and survival benefit. Tumor antigen markers are known to
be increased by the heat shock response and may further enhance
immunologic surveillance. The patient had no history of
thermoregulatory problems but refused to be placed in wet suit or
survival suit because of a "phobia of enclosed tight garments".
It was elected to treat the patient with hyperthermochemotherapy.
Treatment consisted of DNP, and combination chemotherapy with
carboplatin, mitomycin, and doxifluridine. An .alpha.-1 adrenergic
receptor agonist was used to minimize peripheral vascular dilation
and heat loss.
Pretreatment protocol: the patient was transfused with three units
of packed red blood cells. A Foley catheter was inserted on each
day of treatment. She was covered in a water soaked blanket
containing a polyethylene lining. A shower cap with towels was used
to prevent heat loss from the head. Intravenous lines were placed
into both arms with 19 gauge intracaths. EKG, heart rate, rectal
thermistor, and V0.sub.2 monitors were attached. Oxygen
supplemented facemask and equipment was attached and data monitored
as previously described under Example 1.
Treatment protocol: the patient was given chemotherapy by mouth.
The total doses of carboplatin, and mitomycin were 450 mg and 24 mg
IV respectively on day 1 and last day of week 6. Doxifluridine, 600
mg, was orally administered every day for 5 days and repeated the
last 5 days of week 6. On the day of DNP infusion, baseline
recordings were established for 10 minutes. Mephenteramine sulfate,
30 mg, was given by intramuscular injection. Ten minutes later her
heart rate increased to 96 and her V0.sub.2 increased from 250 to
320 cc/min. V0.sub.2, heart rate and blood pressure stabilized
after 20 minutes and she was given an initial dose of 1 mg/kg DNP.
Additional 0.5 mg/kg infusions of DNP were administered in 3
successive infusions spaced 20 minutes apart. The patients V0.sub.2
stabilized between 780-820 cc/min. and her core temperature
increased to a maximum of 41.4.degree. C. After a plateau
temperature of 41.5.degree. C..-+.0.5.degree. C. was reached, her
level of V0.sub.2 and temperature was maintained for a period of 2
hours and 30 minutes with an additional infusion of 0.5 mg/kg DNP
given 50 minutes after the last dose. The DNP treatment protocol
was repeated every fourth day for a period of 6 weeks. A
representative monitored flow chart is shown in FIG. 19.
Treatment outcome: By the combined treatments outlined above,
ascites resolved by the end of the sixth week. Serum levels of
amylase and all tumor markers decreased after the third week of
treatment and were normal at week 6. Repeat magnetic resonance
imaging and echo re-examination of the abdomen showed complete
resolution of peritoneal metastasis. Nine and a half months after
treatment, the patient is alive without any evidence of tumor
reoccurrence.
EXAMPLE 5
Use of DNP with Thermosensitive Liposomes
To overcome the toxicity to normal tissues of many anticancer
agents such as doxorubicin and anti-infectious drugs such as
amphotericin B, liposomal formulations have been developed.
Liposomal doxorubicin is known to have reduced cardiotoxicity and
increased antineoplastic efficacy. Thermosensitive liposomes can
further enhance tumor targeting and decrease toxicity by release of
their water soluble drug contents in response to tumor
hyperthermia. Various synthetic and natural lipids such as
dipalmitoyl phosphatidyl choline and distearoyl phosphatidyl
choline or egg phosphatidyl choline and cholesterol can be combined
in different molar ratios with ethanol, or other agents that have a
biphasic effect on gel-to-liquid phase transition of phosphatidyl
choline bilayers, to produce liposomes that melt (undergo
gel-to-liquid crystalline phase transitions) at a predetermined
hyperthermic temperature.
Thermosensitive liposomes were prepared form phosphatidyl choline
(PC) and cholesterol (Ch) using the ethanol method of Tamura et al.
A combination of PC:Ch in a 8:1 molar ratio in the presence of 6%
(v/v) ethanol resulted in formation of liposomes having a
transition temperature between 40.2 and 40.8.degree. C. The
anticancer drug dacarbazine [5-(3,3'-dimethyl-1-triazino)
imidazole-4-carboxamide] was encapsulated in these heat-sensitive
liposomes at a concentration of 3 mg/ml. The in vivo efficacy of
the thermosensitive, liposome encapsulated dacarbazine was tested
on Swiss albino mice transplanted with a dimethyl
benzo-dithionaphthene derived ascites fibrosarcoma subjected to DNP
induced hyperthermia.
Male, 10-12-week-old, Swiss albino mice were injected with
3.times.10.sup.6 viable fibrosarcoma cells into the peritoneum.
After 15 days the animals were divided into various treatment and
control groups receiving intraperitoneal injections of free
dacarbazine, DNP alone, DNP+empty liposomes and DNP+liposome
encapsulated dacarbazine. DNP induced hyperthermia was recorded
with neonatal rectal and 22 ga. hypodermic YSI probes. Temperatures
were recorded 30 minutes after a 20 mg/kg intraperitoneal dose of
DNP. DNP was administered every day for a total of 5 doses. In all
cases the hypodermic, intraperitoneal temperatures were 1.degree.
C. higher than the rectal.
As shown in FIG. 20, survival curves of animals treated with DNP
alone and DNP+drug containing liposomes were significantly improved
in comparison to controls. DNP-hyperthermia treated animals
remained alive at day 100 whereas sham treated animals all died by
60.
EXAMPLE 6
Use of DNP to Induce Autologous Heat Shock Proteins as a Form of
Thermal Preconditioning Prior to Arterial Balloon Catheterization
or Ischemic Surgical Injury
DNP would be given orally at doses to increase the V0.sub.2 from
1.5 to 5 times above normal per day for a period of 2-6 days or, as
an infusion at doses that would increase V0.sub.2 and core body
temperatures no greater than 39.degree. C. for periods of 5 to 6
hours or, intravenous doses of DNP alone, with vasopressors, or
other short acting metabolic stimulators, that would increase V02
to equivalent core temperatures of 40-41.degree. C. for periods of
15-30 minutes. Within 8-48 hours after cessation of DNP, the
patient would have maximum heat shock protein production. Such DNP
induced stress would improve clinical outcome by induction of
cellular heat shock protein synthesis with protection of the
patient's, organs, tissues and cells from subsequent ischemic
surgical or traumatic procedures.
This method of DNP induced preconditioning could be used to
decrease intimal thickening and restenosis after angioplasty,
improve ischemia/reperfusion injury in organ and tissue
transplantation, and improve surgical outcome of procedures that
require temporary or prolonged occlusion of arterial blood flow.
Examples of such DNP induced autologous thermotolerance used as a
form of preconditioning are depicted in FIG. 21, which shows
limitation of proliferative arterial catheter balloon injury in
Sprague-Dawley rats pretreated with DNP induced hyperthermia; FIG.
22 shows the protective effect of DNP pretreatment before hepatic
ischemic injury cased by Pringle's maneuver; and, FIG. 23 depicts
improved musculocutaneous flap skin survival after induction of
heat shock proteins by DNP.
EXAMPLE 7
Method of Using DNP to Enhance Proton Emission Tomography (Pet) in
the Diagnosis of Malignancy and/or Malignant Transformation
(Glioma)
History: A 24 year old white male with neurofibromatosis presented
with a six month history of left sided loss of body sensation,
emotional changes, sensory seizures, inattention to conversations
and sensations of jamais vu.
Physical examination: weight=65 kg; height=175 cm; BP=135/80; HR=86
& reg; Resp=18; T=37.9.degree. C. The patient was a well
developed well nourished white male with left upper and lower
extremity sensory loss, postural instability and loss of tactile
discrimination. There was a frank left handed astereognosis. Eye
examination was normal, without papilledema.
Laboratory studies: Complete hemogram, blood chemistry and
endocrine examination were normal. EEG was within normal limits.
MRI with gadolinium enhancement showed a decreased signal in the
right temporoparietal region with no evidence of contrast
enhancement. PET examination with
[.sup.18F]fluoro-2-deoxy-D-glucose (FDG) revealed a homogeneous
hypometabolic area (metabolic Grade 1) consistent with a Low grade
glioma in the right temporoparietal region. There were no zones of
high FDG uptake. Differentiation of displaced noninvaded gray
matter from the tumor was not discernible on PET imaging.
Clinical assessment and diagnostic evaluation: although Low grade
gliomas generally present histological features of benign tumor, it
is known that the presence of zones of high FDG uptake by PET scan
in such gliomas is associated with a higher percentage of malignant
transformation. PET-FDG with evidence of tumor hypermetabolism is
believed to be an early biochemical marker of cellular malignant
transformation and is of prognostic value in High grade gliomas.
Biochemically, high glucose (uptake of FDG) utilization in the
presence of oxygen, known as aerobic glycolysis, is believed to be
the result of a hyperactive hexokinase attached to tumor
mitochondria. Increased FDG uptake therefore, represents increased
hexokinase activity and is associated with increased aggressiveness
in gliomas, meningiomas and other neoplasms. Since DNP uncouples
oxidative phosphorylation, any shortfall in mitochondrial ATP
production must come from increased glycolysis. As a result, FDG
uptake will be proportionately increased in DNP treated malignant
cells over those that are normal in contralateral brain white and
gray matter. Since no abnormal FDG uptake was detected in the tumor
by standard PET methodology and the PET scan was unable to clearly
delineate the borders of the tumor, it was elected to give the
patient a low dose of DNP to enhance FDG uptake and repeat the PET
scan. Hypermetabolic components of the tumor would thus permit a
more focused PET-guided stereotactic biopsy.
Pretreatment protocol: three days prior to DNP dosing and repeat
PET-FDG scan, the patient's dosage of phenyloin was increased from
100-mg three times daily to 200-mg three times a day. The same
positron emission tomogram, a CTI-Siemens 933/08-12 which provides
a 6.75-mm adjacent slices and in-plane spatial resolution
(full-width at half maximum) of .about.5 mm, was to be used. The
highest level of non to DNP stimulated FDG uptake in the tumor area
was to be compared and qualitatively graded by two radiologists.
Independently, each investigator was to visually evaluate the
positron emission tomogram and use the following metabolic grading
scale: I, FDG uptake less than contralateral white matter; II,
uptake between the levels in contralateral white and gray matter;
III, FDG uptake equal to or greater than in contralateral gray
matter.
Diagnostic--treatment protocol: the patient was given a 300 mg
capsule of DNP (approximately 4 mg/kg body weight) three hours
prior to undergoing a PET-FDG scan. Forty minutes prior to the
emission scan he was intravenously injected with a bolus of FDG
according to standard methodology. Immediately prior to the
20-minute emission scan the patients VO.sub.2 uptake was 40% above
that at baseline. The patients DNP/VO.sub.2 flow chart is FIG.
24.
Diagnostic outcome: DNP enhanced PET-FDG scan revealed two areas of
hypermetabolism. One of the areas surpassed the limits of the
lesion on CT images and consequently only one of the targets
(graded as a III on FDG uptake) was selected in the "abnormal
PET-normal CT" area.
The plane that best displayed the abnormal FDG hypermetabolic
uptake area was selected and a pixel located in the center of the
zone was interactively pointed at on visual inspection. The
coordinates of that DNP induced hypermetabolic pixel were then
calculated and set as a target for biopsy. A PET-guided
stereotactic biopsy was performed under the procedure described by
Levivier et al., i.e., the target from the PET image was projected
onto the corresponding stereotactic computed tomographic (CT) slice
to control the reliability and precision of target selection and
the trajectory. Serial stereotactic biopsies were performed along
the trajectory by the method described by Kelly et al.
On pathologic examination, including analysis of nuclear
polymorphism and cell density, 2 foci of anaplasia consistent with
glioblastoma (Grade III astrocytoma) were noted.
Treatment outcome: based on the DNP enhanced PET-FDG scan
diagnostics outline above, this patient was found to have a
malignant transformation in his otherwise Low grade glioma. This
diagnostic treatment protocol procedure of detecting foci of
hypermetabolism caused him to undergo systematic radiation therapy
with chemotherapy (dibromodulcitol-procarbazine-carmustine) early
in the course of his malignant process. One year after diagnosis
and therapy the patient again underwent PET scanning. DNP
enhancement (repeated as outlined under "Diagnostic" above)
revealed a single hypermetabolic component (metabolic Grade II) in
the tumor area. Repeat PET-guided biopsy revealed the area to be a
zone of radionecrosis. The remaining viable tumor, even with DNP
enhancement, continued to be a metabolic Grade I. The patient
remains alive one and a half years after his diagnosis, albeit with
left-sided hemiparesis.
EXAMPLE 8
Method of Using DNP to Enhance Detection of Malignant Tumors by
High Resolution Digital Infrared Imaging (Breast Carcinoma)
History: a 34 year old white female with existing fibrocystic
disease of the breast underwent yearly mammography and was found to
have an equivocal opacity in the right breast, medial to the
aereola. Two past breast biopsies were negative for malignancy and
consistent with fibroadenomatous disease of the breast. The patient
was opposed to another breast biopsy (would be third), unless there
was a definitive indication of a lesion over that of her known
fibrocystic disease of the breasts.
Physical examination: WT=60 kg; HT=164 cm; BP=120/72; HR=88 &
reg; R=18/min; T=37.7 C. The patient was a normal appearing white
female with scattered to diffuse nodularities in both breasts. A
palpable 3.times.2 cm, non-tender, lump was located 3 cm medial to
the right aereola.
There was absence of nipple discharge, retraction, skin dimpling,
rash or discoloration of either breast. There were no palpable
axillary lymphadenopathy.
Laboratory studies: chest x-ray, EKG, blood chemistry, and hemogram
examination was normal. Mammography, Doppler ultrasound, MRI, and
scintinammography failed to indicate or eliminate a possible occult
carcinoma in this young patient with dense, fibroadenomatous breast
disease. A diffuse, non-cystic, opacity on the right breast was the
only definitive finding from these breast studies.
Clinical assessment and diagnostic evaluation: this patient has had
two previous open breast biopsies without evidence of malignancy.
Early detection of breast carcinoma is of crucial importance to
survival. False negative results of mammography (and other
complimentary studies) range between 5-30%. The ability of infrared
imaging technology to detect changes related to increased
metabolism (tumor) and angiogenesis has greatly improved from that
of 30 years ago. High resolution digital computerized infrared
equipment can now detect focal increases in tumor temperature from
as little as 0.05.degree. C., and increases in focal breast
temperatures may be as high as 1-2.degree. C. in malignant tumors
versus normal, contralateral breast sites.
Since it is known that infrared imaging has at least a 19% rate of
false positives and 17% of false negatives, and equivocal
mammography and abnormal infrared imaging is not uncommon in young
women with dense breast tissue and diffuse fibrocystic disease, the
use of DNP to enhance tumor metabolism (infrared imaging) over that
of normal tissue, could be of substantial diagnostic benefit.
Specifically, DNP would greatly enhance tumor metabolism (infrared
imaging), in comparison to non-DNP enhanced infrared imaging and
would greatly increase tumor detection when there is either
insufficient production or detection of metabolic heat or vascular
changes. Further, the heat differential between DNP enhanced and
non-DNP infrared tumor imaging may also decrease the false positive
rate seen with this procedure, especially in benign conditions such
as fibrocystic disease of the breast. Since non-DNP infrared
imaging is capable of detecting as great as 1-3.degree. C. changes
in focal temperature between normal and malignant tissue, DNP
enhancement would increase the temperature difference several fold
and enhance both the sensitivity and precision of currently
available infrared imaging technology. The patient agreed to have
both of her breasts examined non-invasively with infrared imaging,
before and after intravenous DNP administration to ascertain if
there was increased infrared signaling from the worrisome, palpable
lump in her right breast.
Prediagnostic protocol: the patient was disrobed to the waist and
sat with her hands interlocked over her head for a five minute
equilibration period in a draft free, thermally controlled
room--kept between 18.degree. C. and 20.degree. C. She did not take
any oral medication, alcohol, coffee, and did not smoke, exercise
or use deodorant three hours prior to testing. A baseline of 4
images consisting of an anterior, undersurface and 2 lateral views
of each breast were generated by an integrated infrared imaging
station consisting of a scanning mirror optical system containing a
mercury-cadmium-telluride detector (Bales Scientific, CA). The
infrared system had a spatial resolution of 600 optical lines, a
central computerized software processor providing multi-tasking
capabilities and a high-resolution color monitor capable of
displaying 1024.times.768 resolution points with 110 colors or
shades of gray per image. Images were stored on retrievable laser
discs.
Diagnostic treatment protocol: after the above baseline studies
were performed, the patient was given an initial intravenous dose
of 1 mg/kg DNP and observed for a period of 20 minutes. An
additional 2 mg/kg of DNP was then administered and 30 minutes
thereafter, she was taken to the thermally controlled room for
repeat DNP-enhanced infrared imaging. Immediately prior to
transferring the patient to the thermally controlled room, the
patients VO.sub.2 was incrementally increased to 50% above her
VO.sub.2 baseline, see FIG. 25. Repeat infrared images were then
obtained under the exact protocol used for obtaining baseline
studies.
Diagnostic--treatment outcome: baseline (non-DNP enhanced) infrared
imaging revealed insignificant vascular asymmetry and no
significant temperature changes when the results were reviewed and
compared to the rest of the ipsilateral or contralateral breast
sites.
DNP enhanced infrared imaging resulted in a bilateral global breast
temperature increase of approximately 0.5.degree. C. An abnormal,
2.5.degree. C. increase in temperature was noted in the palpable,
right breast lesion discovered by clinical exam. Since no
non-cancer causes for such a dramatic temperature increase could be
identified, i.e. abcess, trauma, or recent surgery, this 5 fold
increase in heat production (above the DNP baseline increase of
0.5.degree. C.) was highly suspect to be caused by an early
malignancy.
The patient was admitted to the hospital and under general
anesthesia underwent an open breast biopsy. Frozen section (and
later permanent tissue mounts) revealed a well-differentiated
intraductal carcinoma. Progesterone and estrogen receptors, as
determined by immunocytochemical methods, were negative. A simple,
right mastectomy with axillary lymph node dissection was performed.
A total of twelve lymph nodes were identified: there was no
evidence of tumor. The patient refused chemotherapy and
radiotherapy. She was placed on long-term oral tamoxifen (10 mg
twice a day).
EXAMPLE 9
The Use of Dinitrophenol with Artificial Electron Receptors (or
Other Free Radical Forming Agents) in the Treatment of Hormone and
Chemotherapy Resistant Malignancy (Prostate Cancer)
History: a 68 year old Mexican male, developed a gradual increase
in low back pain, right hip pain and several episodes of hematuria
over a 10 month period. He was referred to a urologist and
diagnostic work-up revealed a carcinoma of the prostate with the
extension of the tumor into the bladder. Bony metastasis were
present to the right pelvis, fourth and fifth lumbar vertebra,
right femur, left humerus, right sixth and seventh ribs and right
scapula. He refused any form of surgery but underwent radiation
therapy to the pelvis and symptomatic bony lesions. Treatment was
initiated with megestrol acetate (640 mg/day), prednisone (20
mg/day) and leuprolide (7.5 mg/month). After three months of
therapy the patient continued to have progression of his disease
manifested by increasing bone pain, rising prostatic specific
antigen levels (PSA) and increasing serum acid phosphatase.
Physical examination: WT=72 kg; HT=175 cm; BP=140/86; R=22; T=37.6
C; HR=88 & reg; Exam revealed mild emaciation with some scrotal
and +1 pitting bilateral lower extremity edema. There were
scattered bilateral, basilar rales on examination of the chest.
Laboratory studies: EKG demonstrated a right partial bundle branch
block. Chest x-ray showed mild chronic obstructive pulmonary
disease with minimal fibrosis. There was some patchy, interstitial
edema in both lower lung fields. There were no pulmonary
metastasis. Complete blood count showed a mild anemia with a
hemoglobin of 10.5 and a hematocrit of 34%. Liver function tests
were normal. White blood cell count, differential and platelet
count, was within normal limits. PSA level was 58 ng/ml. Serum acid
phosphatase was 2.times. above normal. Blood electrolytes including
calcium were within normal limits. The acid phosphatase, AST, ALT
and bilirubin levels were normal. Radionucleotide bone scan
revealed multiple metastasis in the axial skeleton and ribs. Review
of past prostatic biopsy slides showed a high grade adenocarcinoma
of the prostate with a Gleason Grade of 8. Pulmonary function
studies showed moderate airflow obstruction with mild hypoxemia and
hypercarbia. Stress EKG was not performed because of his severe
exercise intolerance.
Clinical assessment and treatment evaluation: the patient has a
metastatic, hormone-refractory prostate carcinoma with clinical
progression documented by increasing bone pain and rising serial
PSA values. Under the TNM classification of the American Joint
Cancer Committee for prostate cancer (T=degree of primary tumor
extension; N=regional lymph node involvement; and, M=presence of
distant metastasis), he has the highest stage (T4 N3 M1).
Histologically, the tumor is aggressive by the Gleason Grading
System. Since death due to prostatic carcinoma is almost invariably
a result of failure to control metastatic disease, and since
prostatic cancers are well-known to be sensitive to heat stress,
the present DNP therapy was undertaken as a last resort effort to
stop tumor progression and/or improve the patients quality of
life.
In view of the patients age, pulmonary problems and poor
performance status (Karnofsky Score of 6) it was decided to treat
the patient with moderate doses of DNP and a free radical cycling
agent, methylene blue (MB), to induce synergistic tumor killing.
The effect of methylene blue on cellular reduction-oxidation status
(redox) is well known. Methylene blue readily traverses cell
membranes and acts as an electron acceptor from the major
coenzymes. Unlike other oxidizing drugs, it cycles futilely,
transferring electrons from endogenous substrates to oxygen.
Depending on the redox status of a cell, MB can act as either an
intracellular electron acceptor or donor. MB directly catalyzes the
reaction of intracellular reductants, NADPH, NADH and GSH (reduced
glutathione) with oxygen causing the production of hydrogen
peroxide, superoxide anions, and the formation of the potent
cytotoxic oxidant species, peroxynitrite. In DNP partially
uncoupled mitochondria, MB further stimulates respiration due to
its dual action of providing reducing equivalents necessary for
beta-oxidation of fats and electron donating/shuttling capacity,
with respect to the mitochondrial respiratory chain. It is an
effective drug, at doses of 1-3 mg/kg, in treating nitrate-induced
methemoglobinemia. MB is also used as an antidote given as a 100 mg
IV bolus for encephalopathy associated with alkylating
chemotherapy.
Since uncoupling, heat and MB increase the flux of cellular free
radicals and malignant cells possess a high bioreductive capacity,
the synergistic effects of DNP with MB would allow for maximum
tumor killing with minimum to moderate levels of induced total body
hyperthermia. Additional free radical cycling agents that can be
used in lieu of MB include, but are not limited to: phenazine
methosulfate, xenobiotics such as quinones (e.g., menadione,
semiquinone, naphthoquinone, duroquinone, indigo carmine),
nitrocompounds (e.g., metronidazole, niridazole, nitrofurazone,
flunitrazepam), eminium ions (e.g., methyl viologen, benzyl
viologen, etc.), and others. In this patient, DNP-MB therapy was to
be administered so as not to exceed the baseline VO.sub.2 level by
50-75%.
Pretreatment protocol: the patient was transfused with 2 units of
packed red blood cells 48 hours prior to undergoing treatment.
Intravenous fluids (Lactated Ringer's solution) were administered
at a rate of 100 cc/hour. The patient was dressed in comfortable
cotton clothing and placed in an air-conditioned room. Equipment
for monitoring heart rate and rhythm, temperature and oxygen
consumption was utilized as outlined in Example 1. An oral
breathing tube was used to conduct TEEM VO.sub.2 measurements.
Oxygen supplementation and "crash cart" was available at
bedside.
Treatment protocol: baseline VO.sub.2 measurements for 8 minutes
established an average VO.sub.2 of 250 cc/minute. DNP, at a dose of
2 mg/kg, was infused intravenously over a 2 minute period. Repeat
VO.sub.2 at 20 minutes was stabilized at 340-360 cc/minute. An
additional 1 mg/kg DNP infusion was administered, and 15 minutes
thereafter the VO.sub.2 increased and stabilized at 420 cc/minute.
Ten minutes thereafter, an infusion of methylene blue, 2 mg/kg
(dissolved in a 0.4% pyrogen-free isotonic saline solution-35 ml)
was administered over 20 minutes. Repeat VO.sub.2 measurement at 20
minute intervals showed it to rise to and stabilize at 450-500
cc/minute.
By hour 3, VO.sub.2 declined to the 360-380 cc/minute range. An
additional 1 mg/kg dose of DNP was infused over a 2 minute period.
Repeat VO.sub.2 measurements 20 minutes after this infusion showed
an increase in VO.sub.2 back to the 450-500 cc/minute. Rectal probe
temperature increased to a maximum of 1.3.degree. C. over baseline.
Blood pressure and cardiac rates remained within normal limits. The
patient withstood the procedure without any adverse effects and
therapy was terminated 6 hours after the initial DNP dose. The
protocol was repeated every other day for a total of 15 treatments
(30 days). Therapy was discontinued for 2 weeks and the cycle was
again repeated for an additional 30 days, treatment being
administered every other day.
Treatment outcome: there was no evidence of general toxicity at any
time during treatment. The patient noted a decrease in his low
back, hip and other areas of bone pain on the 6.sup.th day
following therapy. By 2 weeks, the patient was off all narcotic
(morphine) analgesics and had a markedly increased appetite. On day
8, repeat PSA levels were increased by approximately 120% to 125
ng/ml. Acid phosphatase remained unchanged. All other blood
chemistries, including CBC, showed no significant alterations.
At 6 weeks after treatment, repeat PSA values showed a significant
decline to 30 ng/ml with a concomitant fall in serum acid
phosphatase levels. At the final stage, 10 weeks after initiation
of treatment, a prostatic biopsy was performed. Histologic
examination revealed 95% of the tumor to be necrotic with only
scattered or scarred acini containing an occasional malignant cell.
There was a significant increase in stromal cells above that seen
in his initial biopsy. One of the most striking changes noted by
the pathologist was the formation of cyst-like structures within
the epithelial cells. The patient was seen three months after
initiation of therapy, at which time he had gained 8.2 kg of
weight, remained pain free and stated that he felt "normal". FIG.
26 shows monitored treatment parameters. FIG. 27 shows biochemical,
biopsy and clinical responses.
Oral DNP therapy (250 mg twice a day, daily for 5 days and recycled
after no medication for 2 days) was initiated after his IV therapy
and continued up to 4 months. A repeat prostate biopsy at the end
of month 4 was obtained. Pathologic examination revealed
disintegration of remaining tumor acini along with the formation of
with many epithelial cysts. Occasional residual tumor cells were
fractured and disrupted with markedly reduced cytoplasm. There was
extensive fibrosis with an apparent increase in the number of
stromal cells. Cytoplasm volume was significantly diminished in
both the residual tumor and normal cells. Overall, there were very
few intact acini or viable acinar cells.
EXAMPLE 10
Method of Using Dinitrophenol with Biologic Response Modifiers (in
the Treatment of Hepatitis C Infection)
History: a 32 year old Investment Banker was evaluated for chronic
Hepatitis C infection. She gave a past history of intermittent
jaundice, dark urine, mild anorexia, nausea and vomiting. This
episode occurred 10 years ago, approximately 3 months after a
transfusion (3 units of packed red blood cells) for a cesarean
section. She was currently asymptomatic, but on routine health
insurance exam she was found to have elevations in her ALT and AST
(alanine and aspartate aminotransferase) levels: 140 IU/L and 90
IU/L, respectively. She drank 5-8 glasses of wine, per week.
Additional laboratory tests identified anti-HCV antibodies with an
HCV-RNA level of 5.times.10.sup.6/ml. The patient refused to
undergo liver biopsy but agreed to treatment with interferon
alpha-2b (3 million units injected subcutaneously 3 times per week)
and ribavirin (500 mg orally--twice a day). After 12 weeks of
treatment she developed lethargy, severe headaches, fever, nausea
and depression. Anemia was detected with a hemoglobin concentration
of 9.2 g/deciliter. As a result, her dosage of interferon was
reduced to 1.5 million units 3 times a week and the dose of
ribavirin was reduced to a total of 600 mg/daily. After 6 months of
treatment her ALT and AST levels became normal and HCV-RNA became
undetectable.
An additional six months of therapy however, failed to sustain her
clinical improvement and she was found to have a relapse. Serum
HCV-RNA levels rose to 5.2 million copies/ml and liver enzymes
increased to 2.5-3 times that of the normal range. She was unable
to tolerate any additional ribavirin because of severe anemia. She
persistently refused to undergo a percutaneous liver biopsy.
Physical examination: WT=48 kg; HT=150 cm; BP=128/82; HR=76 &
reg; R=18; T=37.5.degree. C. Physical examination failed to reveal
any signs of chronic liver disease. She was noted to have several
scattered areas of scalp alopecia which she attributed to her
anti-hepatitis C therapy.
Laboratory studies: EKG and chest x-ray were normal. CBC revealed a
mild anemia with a hemoglobin of 10.2 and a hematocrit of 34%. WBC,
differential and platelet count were within normal limits. Alkaline
phosphatase was within normal limits. Serum AST and ALT were
elevated to 2.5-3 times that of the upper normal limit. Serum
HCV-RNA levels were found to be at 5.8 million copies/ml. The
infecting hepatitis C strain was of genotype 1b. Antimitochondrial
antibody serology was negative (titer less than 1:20). There were
no other blood chemistry, hormone, or urine laboratory
abnormalities.
Clinical assessment and treatment evaluation: the patient has a
chronic Hepatitis C infection with relapse after combination
ribavirin and interferon alpha-2b treatment. This is not uncommon
in that the rate of relapse after an end-of-treatment response to
interferon-ribavirin therapy may exceed 50%. She was unable to
tolerate additional ribavirin therapy because of a related
anemia.
Further, interferon dose escalation in non-responders to initial
interferon therapy has only proved successful in a small number of
cases. Despite her refusal to undergo any form of liver biopsy she
agreed to undergo a combination of DNP and interferon therapy for a
period of 12 weeks.
The liver is known to be one of the "hottest" organs in the human
body. Liver temperatures exceeding 44.degree. C. have been
documented in humans undergoing strenuous exercise. The hepatitis C
virus is an RNA encoded sphere containing several polyproteins
comprising a capsid, 2 envelope proteins, and at least 6 enzymatic
proteins with varied functions. Hepatitis C virus is known to be
heat sensitive and is inactivated by standard blood banking heating
techniques. Case reports of hepatitis C inactivation with the use
of extracorporeal hyperthermia are known. It has been reported that
HIV positive patients treated with extracorporeal hyperthermia,
many of which were also positive for hepatitis C, the hepatitis C
virus was cleared (as determined by serum viral PCR-RNA
analysis).
Based on the this patients failure to respond to conventional
treatment, anecdotal and case report studies showing beneficial
results with whole body hyperthermia, the patient underwent a
combination of DNP and interferon therapy. She was informed that
she would undergo daily treatments with intravenous DNP for five
days per week and receive interferon alpha at a dose of 1.5 million
units subcutaneously every two days. This treatment protocol would
continue until her hepatitis C-RNA blood viremia was no longer
detectable.
Pretreatment protocol: each evening prior to treatment the patient
was instructed not to eat after 7 pm and dress in cotton clothes.
Approximately 6 hours prior to intravenous DNP administration she
was to be given 1.5 million units of subcutaneous interferon-alpha
every 3rd day. Repeat blood work, including CBC and platelet count,
AST, ALT, and hepatitis C-RNA levels would be initially obtained at
48 hours and weekly thereafter. No efforts were to be made to
prevent body heat loss. A single intravenous line was placed with a
21-gauge interacath. Heart rate, rectal thermistor, and VO.sub.2
monitoring was conducted during therapy as outlined.
Treatment procedure: the patient presented herself for outpatient
treatment and was given a subcutaneous dose of 1.5 million units of
interferon-alpha. Approximately 6 hours thereafter, at 1 pm, a
baseline VO.sub.2 recording of 5 minutes was 160 cc/min. She was
infused with 1 mg/kg DNP over a 2 minute period. At 20 minutes, her
VO.sub.2 increased and stabilized at approximately 210 cc/min. A
second dose of 1 mg/kg DNP was infused and the VO.sub.2 peaked 20
minutes later at 250 cc/min. An additional dose of 2.0 mg/kg DNP
was given 30 minutes following the second dose. Repeat VO.sub.2
showed a rise and stabilization 20 minutes thereafter at 360
cc/min. The patient's rectal temperature increased and never
exceeded 1.3.degree. C. above her normal baseline. Two hours after
her last dose of DNP, her VO.sub.2 declined to 280 cc/min. An
additional 2 mg/kg dose of DNP was administered. The patients
VO.sub.2 increased and stabilized 20 minutes thereafter to a level
of 420 cc/min. She was noted to sweat profusely. Throughout
treatment the patient was permitted to drink fluids ad libitum. She
was notably fatigued at hour 5 of therapy. Monitored parameters and
flow chart are shown in FIG. 23. The 5 day treatment protocol was
repeated after a 2 day "DNP rest period". This regimen was repeated
times 3. Subcutaneous interferon-alpha was administered for a total
of 10 weeks. FIG. 28 shows the patients DNP/interferon treatment
flow chart.
Treatment outcome: by the treatment regimen outlined above,
hepatitis C-RNA viral load decreased by approximately 2 logs after
48 hours. Over the next 5 days the viral load further decreased by
an additional log. HCV-RNA became undetectable and the HCV viral
genome remained cleared from the bloodstream at week 2 and
thereafter. Alanine transaminase (ALT) levels increased 7 fold at
48 hours and remained elevated until week 3, at which time they
returned to levels slightly below that which existed prior to
therapy. CBC, bilirubin, and blood urea nitrogen (BUN) remained
within normal limits. Alkaline phosphatase levels increased 2 fold
at 48 hours but returned to pretreatment levels at day 7.
The patients HCV viral genome remained cleared from her bloodstream
18 months after therapy and there was normalization of her ALT.
EXAMPLE 11
Method of Using Dinitrophenol Induced Intracellular Hyperthermia to
Increase Immunogenicity of Human Tumors
DNP would be given as an intravenous solution, or as an oral
preparation, so as to increase oxygen consumption 2.5-5 times above
normal for a period of 2-3 hours. Such treatment would be
administered every other day for a period of 5-10 days. At 8-24
hours after the last day of treatment, the patient would be
administered standard chemotherapy or specific monoclonal antibody
immunotherapy directed against known mutated or inappropriately
expressed oncogenic proteins (e.g., ras, p53, HER/neu, etc.), or
combination anti-oncogenic immunotherapy with chemotherapy or
radiation.
Heat shock proteins (HSPs) or stress-induced proteins are
constitutively expressed in all living cells and are among the most
abundant proteins found. However, many members of the HSP family
can further be expressed by cellular stress-causing conditions such
as heat, drugs, glucose deprivation, etc. Of importance to the
present method is that the expression of HSPs in tumors is
associated with a heightened immune and/or cytotoxic T-lymphocyte
response. In particular, it is known that members of the HSP70
family (HSPs are generally classified by their molecular weights
e.g., HSP90 kdaltons, HSP27 kdaltons, HSP70 kdaltons, etc.) are
expressed on cell surfaces. Due to the ability of DNP to induce
intracellular hyperthermia, the enhanced expression of human HSPs
in DNP treated tumors could greatly increase their
immunogenicity.
This method could be used to broaden the antigen-specific
repertoire of many poorly immunogenic tumors by increasing the
expression of HSP-peptide immunogenic determinants on their cell
surfaces. Such consequences would heighten any endogenous specific
anti-tumor immune response. Moreover, DNP-intracellular
heat-inducible immunogenic targets could further increase the
efficacy of exogenously synthesized and administered monoclonal
antibodies. By example, patients with HER-2/neu overexpressing
metastatic breast cancer (25% of breast cancer patients) would be
treated by the DNP method outlined above. This treatment would then
be followed by a standard loading dose and weekly infusions of
anti-HER-2/neu monoclonal antibodies. Clinical benefits would be
evaluated by overall response rates and duration of response.
EXAMPLE 12
Synthesis and Use of Novel Conjugates and Derivatives of
2,4-Dinitrophenol
Formation of novel nitrophenol compounds is of importance in that
their alkyl, alkene, fatty acid, aromatic and other derivatives may
significantly enhance their biologic activity and/or improve the
therapeutic index. Many reactions of the benzene ring of phenols
through halogenation, sulfonation, and nitration are known.
Numerous procedures for C-alkylation of phenols through reduction
of benzylic alcohol, aldehydes, benzonitriles and Mannich bases are
published.
Alkylations or other "R" group additions have also been performed
on various phenolic substrates using Stille or Negishi coupling
reactions. An example of converting a nitrophenol compound to the
desired alkylated (or other "R" group analog) by a 2 step procedure
utilizing the Stille coupling reaction is illustrated in FIG. 30.
As shown in step 1, DNP is first iodinated with Barluenga's reagent
(IPy.sub.2BF.sub.4) to yield 2,4-dinitro-3,5-diiodophenol. In step
2, the nitroiodophenol is then converted to the alkylated
derivative (in the instant example an ethylated derivative) via a
co-catalytic, palladium-copper Stille reaction.
Compound 3 shown in FIG. 30 is an ethylated derivative of DNP and
is designed to increase uncoupling activity by adding lipophilic
alkyl substituents to the benzene ring. Such analogs with augmented
activity may be particularly useful in the treatment of bulky
tumors and malignancies which possess a high fat content, e.g.
liposarcoma, glioblastoma, etc.
A representative approach (Step 2) to the palladium-copper,
co-catalytic ethylation of a nitroiodophenol is illustrated by the
conversion of 2,4-dinitro-3,5-diiodophenol to
2,4-dinitro-3,5-diethylphenol. Nitroiodophenol (500 mg, 9341 mmol)
is added to a pressurized reaction to containing
N-ethylpyrrolidinone (1.5 ml). Pd.sub.2dba.sub.3CHCL.sub.3 (27 mg,
26 .mu.mol) and triphenylphosphine (50 mg, 191 .mu.mol) is added to
the stirring solution and slowly heated to approximately 50.degree.
C. for 10 minutes. Copper iodide (17 mg, 91 .mu.mol) is added to
the stirring solution. The mixture is again heated to 50.degree. C.
for 10 minutes. The solution is cooled to 32.degree. C. and
tetraethyl tin (285 .mu.L, 2.05 mmol) is added to the stirring
solution. The reaction tube is sealed and heated with continuous
stirring at 65.degree. C. for 12-16 hours. Aqueous workup and ethyl
acetate extraction with drying by magnesium sulfate (MgSO.sub.4)
and concentration yields the end product.
EXAMPLE 13
Synthesis of an Expanded Combinatorial Library of Putative
Uncoupling Agents Capable of Inducing Intracellular
Hyperthermia
The spectrum of potential classic uncoupling agents that can induce
intracellular hyperthermia can be greatly expanded through a
designed convergent synthetic approach. An almost limitless variety
of uncouplers can be synthesized through a "combinatorialized"
scheme to produce an expanded "library" of uncoupling agents with
related structures. The scheme specifically presented herein
exemplifies the synthesis of 21 potential uncoupling agents, but
can be expanded to 1,000 to 100,000 putative uncoupling agents.
Five classes of uncouplers are prepared via the convergent route
shown in FIG. 31. The synthetic scheme depicted in FIG. 31 is
designed as a combinatorial approach to allow access to a library
of structurally related putative uncouplers for biological
evaluation. While the given examples noted in FIG. 31 will allow
formation of at least 21 novel uncouplers, a simple variation in
this synthetic scheme will allow the library of uncouplers to be
expanded to include from 1,000 to 100,000 novel uncoupling agents.
After discussing the general synthetic approach in FIG. 31, the
simple synthetic variations designed to expand the library of
uncouplers will be described. Such variations will be apparent to
those skilled in the art of synthetic organic chemistry and
pharmaceutical development.
Starting from benzaldehyde (FIG. 31, Compound 1), diiodination at
the 3- and 5-positions using Barluenga's reagent
(IPy.sub.2BF.sub.4) affords Compound 2 which is alkylated using a
co-catalytic, palladium-copper Stille reaction to produce a
3,5-disubstituted Compound 3. This 2 step approach is known for
producing a variety of methylated phenols. Use of tetramethyltin
then produces the dimethyl derivatives [Compound 3, where R=Me
(methyl)]; tetrabutyltin produces the dibutyl derivatives [Compound
3, where R=Bu (butyl)]; and, tetraphenyltin produces the diphenyl
derivatives [Compound 3, where R=Ph (phenyl)]. A Baeyer-Villiger
oxidation of Compound 3, with meta-chlorobenzoic peracid (mCPBA)
followed by alkaline hydrolysis [KOH (potassium hydroxide)] of the
resulting formate affords phenols, Compound 4. The homogeneous
2,4-dinitro- or 2,4-dicyano-derivatives are initially accessed from
an intermediate Compound 4. Nitrosation of Compound 4 with
nitrofluoromethylsulfonate salt (NO.sub.2CF.sub.3SO.sub.3) provides
the 3,5-disubstituted-2,4-dinitrophenols shown in Compound 5. Three
different uncoupling agents are produced via this synthetic route.
Diiodination of Compound 4 at the 2- and 4-positions produces
Compound 6 which is treated with copper(I) cyanide (CuCN) to give
the 2,6-dicyanate derivative, Compound 7. Three additional
uncouplers are synthesized by this route. The heterogeneous nitro-,
cyano-uncouplers are also accessed from intermediate Compound 3.
The 2-cyano-, 4-nitro-uncouplers are targeted as the steric effects
of the cyano group at the 2-position is less than the corresponding
2-nitro-derivatives. Mono-iodination of Compound 3 through the
thallium intermediate affords the selective 2-iodo-derivative,
Compound 8. Conversion of Compound 8 to phenol, Compound 9, is
accomplished as before through the Baeyer-Villiger oxidation and
hydrolysis of the resulting formate. Selective 4-nitration to
produce Compound 10 is readily accomplished with
nitrotrifluoromethylsulfonate salt followed by cyanation to afford
2-cyano-, 4-nitro-uncouplers, Compound 11. Three additional
uncouplers are produced by this route.
Additional uncouplers, such as the 2,4,6-tricyano compounds, can
also be produced through the same convergent synthesis. Exhaustive
iodination of Compound 4 affords 2,4,6-triiodinated Compound 12
which is then directly converted to tricyano-uncouplers, Compound
13, through copper catalyzed exchange. Three more uncouplers are
produced by this modification. A 2,4-dicyano-uncoupler carrying
three variable substituents at the 3-, 5- and 6-positions is also
readily produced through this convergent approach. Initial
selective monobromination of the phenol Compound 4 at the
ortho-position affords Compound 14 which is diiodinated at the
2,4-positions to produce the 2,4-diiodo-, 6-bromo-Compound 15
derivatives. Selective cyano exchange at the more reactive
aryliodide positions affords the dicyano Compound 16 derivatives. A
final co-catalytic, palladium-copper Stille reaction results in the
formation of the 3,5,6-trisubstituted, 2,4-dicyano-uncouplers. Use
of the same tin reagents previously described allows the
introduction of either methyl, ethyl, propyl, butyl, etc., or
phenyl at the 6-position. In conjunction with the 3 different
substituents at the 3- and 5-positions, 9 additional uncouplers are
afforded by this additional expansive route.
The synthesis of 21 novel uncouplers depicted by the convergent
approach in FIG. 31 can be further modified. To those skilled in
the art, a simple variation in this exemplary synthetic approach
will allow a greatly expanded library of potential uncouplers to be
synthesized. The expanded library can be produced by introduction
of an array of alkyl and aryl substituents at the 3-, 5-, and/or
6-positions while maintaining the 2,4-dinitro-, 2,4-dicyano,
2-cyano-4-nitro-, and/or the 2,4,6-tricyano-phenol substrate. This
simple synthetic variation is accomplished by using a variety of
well known palladium, zinc, or copper-mediated reactions at the
stage of alkyl or aryl group incorporation, i.e., FIG. 31, Compound
2 to 3 and Compound 16 to 17 conversions. This synthesis is a
variation on the Stille reaction, the Heck reaction, the Negishi
coupling, Suzuki couplings, Semmelhack reactions and cuprate
reactions. Such a variation can introduce a nearly of unlimited
array of potential substituents onto the phenol core of the
uncoupler. This combinatorial approach can even be further expanded
by variable halogenation (either bromination or iodination) at the
3- and 5-positions to allow 2 different substituents to be
introduced at these 2 positions in the ensuing metal-mediated
halogen exchange reactions. This "combinatorial library" approach
will allow a broad range of potential uncouplers to be synthesized
and evaluated for potential biological activity, including safety
and effectiveness.
Activity of the many diverse conjugates and derivatives of
2,4-dinitrophenol (and other uncoupling agents) may be tested by
known in vitro methods for oxygen consumption, e.g., tissue or
cellular suspensions with Clark type oxygen sensors. Toxicity,
mutagenicity and LD50 studies in animals would be performed under
recognized protocols prior to use of any such novel compounds in
human subjects. Upon establishing toxicity and safety criteria, the
various novel conjugates and derivatives can be administered under
dose escalation trials as outlined previously for the clinical use
of dinitrophenol.
It will be apparent to those skilled in the art that numerous
modifications and variations can be made to the processes and
compositions of this invention. Thus, it is intended that the
present invention cover the modifications and variations of this
invention provided they come within the scope of the appended
claims and their equivalents.
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